Multiplex detection compositions, methods, and kits

ABSTRACT

The present invention generally relates to the detection of analytes, particularly biomolecules in samples. The invention also relates to compositions, methods, and kits for detecting the presence of analytes, typically in multiplex detection formats. The invention also relates to methods for determining the presence of at least one analyte in a sample, the methods employing employ single molecule detection techniques to individually detect at least one molecular complex or at least part of a molecular complex.

RELATED APPLICATIONS

This application is related to co-filed patent applications“Compositions, Methods, and Kits for Assembling Probes Comprising CodedMolecular Tags” (U.S. “Express Mail” mail labeling number: EL 897 623158 US) and “Compositions, Methods, and Kits for Fabricating CodedMolecular Tags” (U.S. “Express Mail” mail labeling number: EL 897 623161 US).

INTRODUCTION

Disclosed herein are compositions, methods, and kits for detecting thepresence of analytes in a sample, typically in multiplex detectionformats using single molecule detection techniques (SMDs). Variousqualitative and/or quantitative assay methods are currently used foranalyte analyses such as genotyping, gene expression profiling, forensicidentification, antibody and antigen detection, protein profiling, andother protein and nucleic acid measurements. Such methods typically relyon probes, such as oligonucleotides, antibody molecules orimmunoreactive fragments of antibody molecules, peptides, ligands orreceptors, and the like. These probes are generally labeled with asingle species of label, such as a fluorophore, radioisotope, or enzyme.The label is usually detected in an ensemble measurement, for example, amultitude of labeled molecules are collectively identified and/orquantified.

Multiplex assays typically involve simultaneous or near-simultaneousidentification and/or quantitation of multiple targets in a singlesample or a single pooled sample. While generally decreasing the timeneeded to evaluate multiple targets, such multiplex assays can belimited by the number, availability, and cost of differently labeledprobes used in the assay. Conventional multiplex assays include, forexample, fixed array formats such as nucleic acid microarrays andprotein microarrays, and various bead-based formats. Bead-basedmultiplex assays reportedly provide the benefit of increasedhybridization kinetics compared to fixed arrays, but the use of beadssignificantly increases the cost of these assays.

SUMMARY

Compositions, methods, and kits for determining the presence of at leastone analyte in a sample, including multiplex analyses of multipleanalyte species in one or more samples, are disclosed herein. In certainembodiments, analytes include, for example but are not limited to,proteins; peptides; nucleic acids, including DNA and/or RNA molecules;small molecules; drugs and drug metabolites.

According to certain methods, molecular complexes, diagnostic for thepresence or absence of an analyte in a sample, are formed. Molecularcomplexes typically comprise at least one coded molecular tag thatincludes multiple reporter group species in an ordered pattern.Typically, the multiplicity of reporter group species in a molecularcomplex or at least part of a molecular complex are detected as acoupled assembly, either simultaneously or near-simultaneously, similarin some respects to reading a product identification bar code, but at amolecular level. At least one molecular complex is individually detectedusing at least one SMD to identify the order of the reporter groupspecies in at least one coded molecular tag. In certain embodiments,only part of the molecular complex is individually detected.

In certain embodiments, methods for determining the presence of at leastone analyte in a sample comprise: combining the sample with at least oneprobe set for the at least one analyte, the probe set comprising (a) atleast one first probe comprising at least one first reaction portion and(b) at least one second probe comprising at least one second reactionportion. At least one probe in at least one probe set further comprisesat least one identity portion comprising at least one coded moleculartag. In certain embodiments, at least one first probe and at least onecorresponding second probe are suitable for forming a molecular complexin the presence of at least one corresponding analyte or at least onecorresponding analyte surrogate. When a molecular complex, or at least apart of a molecular complex, is individually detected, the presence ofthe corresponding analyte can be determined by identifying the order ofreporter group species in the molecular complex or at least part of amolecular complex. Conversely, the lack of a particular molecularcomplex indicates that the corresponding analyte is not present in thesample.

In certain embodiments, at least one analyte is amplified forming atleast one amplification product, typically an analyte surrogate. Incertain embodiments, at least one molecular complex comprises at leastone analyte surrogate or at least a part of at least one analytesurrogate and at least one probe comprising at least one identityportion. In certain embodiments, at least one molecular complexcomprises the complement of at least one analyte surrogate or thecomplement of at least a part of an analyte surrogate and at least oneprobe comprising at least one identity portion.

In certain embodiments, at least one analyte, at least part of at leastone analyte, or their complements, are amplified before, during, orafter molecular complex formation. In certain embodiments, the methodsand kits further comprise at least one polymerase, at least one ligationagent, or at least one polymerase and at least one ligation agent. Incertain embodiments, methods comprise ligation reactions; primerextension or “gap filling” reactions; transcription, including but notlimited to reverse transcription; translation; or combinations thereof,including but not limited to, coupled in vitro transcription/translationsystems.

In certain embodiments, individually detecting comprises SMD, including,but not limited to, scanning probe microscopy techniques and appliedoptical spectroscopy techniques. In certain embodiments, at least onemolecular complex or at least a part of a molecular complex becometethered or attached, directly or indirectly, to a substrate by one ormore attachment points. In certain embodiments, at least one molecularcomplex or at least part of a molecular complex is individually detectedwhile interacting with, or being tethered or attached directly orindirectly to, a substrate. In certain embodiments, at least onemolecular complex or at least one part of a molecular complex isindividually detected in solution.

Compositions, methods, and kits for assembling probes are also provided.In certain embodiments, probes comprise at least one reaction portionand at least one identity portion including at least one coded moleculartag. In certain embodiments, probes further comprise at least onecapture ligand, at least one cleavable component, at least onecrosslinker, at least one adapter, or combinations thereof. In certainembodiments, probes are assembled using coded molecular tags andoligonucleotides comprising sequences complementary to target sequencesin at least one analyte, at least one analyte surrogate, or both. Incertain embodiments, probe assembly comprises at least template, atleast one ligation template, or both. In certain embodiments, probes areassembled using coded molecular tags and antibodies thatimmuno-specifically react with at least one analyte, at least oneanalyte surrogate, or both. In certain embodiments, probes are assembledusing coded molecular tags and binding proteins or binding peptides thatbind to at least one analyte, at least one analyte surrogate, or both.In certain embodiments, probes are assembled using coded molecular tagsand aptamers that bind to at least one analyte, at least one analytesurrogate, or both

In certain embodiments, probes sets comprise at least one first probecomprising at least one first reaction portion and at least one secondprobe comprising at least one second reaction portion. At least oneprobe in the probe set further comprises at least one identity portioncomprising at least one coded molecular tag. In certain embodiments,probe sets further comprise at least one capture ligand, at least onehybridization tag, at least one aptamer, at least one mobility modifier,at least one analytical portion, or combinations thereof. In certainembodiments, at least one analytical portion comprises at least onereporter group. In certain embodiments, the reaction portion of at leastone first probe comprises at least one reporter group, the reactionportion of at least one second probe comprise at least one reportergroup, or both. In certain embodiments, the reaction portion of at leastone first probe comprises at least one fluorescent reporter group, thereaction portion of at least one corresponding second probe comprises atleast one fluorescent reporter group, or both, wherein the fluorescentreporter groups are the same or different.

Compositions, methods, and kits for fabricating coded molecular tags arealso provided. In certain embodiments, at least one coded molecular tagis fabricated from subunits, including without limitation, syntheticoligonucleotides, nucleotide fragments, semi-synthetic sequences, orcombinations thereof. In certain embodiments, at least one subunit isenzymatically-labeled with at least one reporter group,chemically-labeled with at least one reporter group, synthesized (e.g.,solid-phase synthesis or template-directed synthesis) with at least oneincorporated reporter group, or combinations thereof. In certainembodiments, compositions, methods, and kits for fabricating at leastone coded molecular tag comprise at least one template, at least oneligation template, or both. In certain embodiments, compositions,methods, and kits for fabricating coded molecular tags comprise at leastone PNA, at least one pcPNA, or both.

In certain embodiments, coded molecular tags further comprise at leastone adapter, at least one crosslinker, or both. In certain embodiments,the coded molecular tag adapter or crosslinker, or both, are cleavable.In certain embodiments, at least one coded molecular tag furthercomprises at least one capture ligand, at least one hybridization tag,at least one aptamer sequence, or combinations thereof. In certainembodiments, at least one coded molecular tag is used to prepare atleast one probe.

Kits for determining the presence of at least one analyte in a sample;kits for assembling at least one probe; and kits for fabricating atleast one coded molecular tag; are also provided. Kits serve to expeditethe performance of the methods of interest by assembling two or morecomponents required for carrying out the methods. Kits generally containcomponents in pre-measured unit amounts to minimize the need formeasurements by end-users. Kits preferably include instructions forperforming one or more methods of the invention. Typically, the kitcomponents are optimized to operate in conjunction with one another. Incertain embodiments, kits comprise at least one probe, at least oneprobe set, or both. In certain embodiments, kits comprise at least oneligation agent; at least one polymerase; at least one nucleotide; atleast one amino acid; at least one charged tRNA; at least one substrate;at least one of reporter group; or combinations thereof.

Certain embodiments of the disclosed methods and kits comprise at leastone ligation agent. In certain embodiments, the ligation agent comprisesat least one ligase, such as DNA ligase or RNA ligase, including,without limitation, the bacteriophage T4 (T4) DNA ligase, T4 RNA ligase,E. coli DNA ligase, or E. coli RNA ligase. In certain embodiments atleast one ligase comprises at least one thermostable ligase. Exemplarythermostable ligases include without limitation, Taq ligase, Pfu ligase,Tfl ligase, Tli ligase, Tth ligase, and the like.

In certain embodiments, ligation is performed non-enzymatically. Whilenot limiting, non-enzymatic ligation includes chemical ligation, suchas, autoligation and ligation in the presence of an “activating” and/ora reducing agent. Non-enzymatic ligation can utilize specific reactivegroups on the respective 3′ and 5′ ends of the probes to be ligated.Thus, in certain embodiments of the methods and kits of the invention,the ligation agent is an “activating” or reducing agent. In certainembodiments, one or more probes suitable for ligation are provided thatcomprise appropriate reactive groups for non-enzymatic ligation.

In certain embodiments the disclosed methods and kits further compriseat least one polymerase, including, but not limited to at least one DNApolymerase, at least one RNA polymerase, at least one reversetranscriptase, or combinations thereof. Exemplary polymerases includeDNA polymerase 1, T4 DNA polymerase, SP6 RNA polymerase, T3 RNApolymerase, T7 RNA polymerase, AMV reverse transcriptase, M-MLV reversetranscriptase, and the like. In certain embodiments, at least one DNApolymerase lacks 5′->3′ exonuclease activity, for example, but notlimited to Klenow fragment of DNA polymerase, 9°N_(m)™ DNA polymerase,Vent_(R)® (exo⁻) DNA polymerase, Deep Vent_(R)® (exo⁻) DNA polymerase,Therminator™ DNA polymerase, and the like. In certain embodiments, atleast one polymerase is thermostable. Exemplary thermostable polymerasesinclude Taq polymerase, Tfl polymerase, Tth polymerase, Tli polymerase,Pfu polymerase, AmpliTaq Gold® polymerase, 9°N_(m)™ DNA polymerase,Vent_(R)® DNA polymerase, Deep Vent_(R)® DNA polymerase, UlTmapolymerase, and the like.

The skilled artisan will understand that any of a number of polymerasesand ligases could be used in the methods and kits of the invention,including without limitation, those isolated from thermostable orhyperthermostable prokaryotic, eukaryotic, or archael organisms. Theskilled artisan will also understand the terms “ligase” and “polymerase”include not only naturally occurring enzymes, but also recombinantenzymes; and enzymatically active fragments, cleavage products, mutants,or variants of such enzymes. Descriptions of ligases and polymerases canbe found in, among other places, Twyman, Advanced Molecular Biology,BIOS Scientific Publishers (1999); Enzyme Resource Guide, rev. 092298,Promega (1998); Sambrook and Russell, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, 3d ed. (2001)(“Sambrook and Russell”);Sambrook, Fritsch, and Maniatis, Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Press, 2d ed. (1989)(“Sambrook et al.”); Ausbel etal., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1995, including supplements through the August 2003)(“Ausbel et al.”).

In certain embodiments, kits comprise at least one coded molecular tag;at least one crosslinker, including without limitation at least onechemical crosslinker, at least one photo-activated crosslinker, at leastone cleavable crosslinker; at least one antibody, including withoutlimitation at least one reporter group-labeled antibody; at least onebinding protein, at least one binding peptide, or both; at least onecapture ligand; at least one capture moiety; at least one hybridizationtag; at least one mobility modifier; at least one aptamer; at least onetemplate, at least one ligation template, or both; or combinationsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: depicts a schematic overview of exemplary embodiments. FIG. 1Adepicts exemplary probes and methods for determining the presence ofnucleic acid analytes. FIG. 1B depicts exemplary probes and methods fordetermining the presence of non-nucleic acid analytes.

FIG. 2: schematically depicts an exemplary molecular complex fordetermining the presence of a nucleic acid analyte.

FIG. 3: depicts schematic representations of exemplary molecularcomplexes.

FIG. 4: schematically depicts several illustrative molecular complexes,each comprising an identity portion comprising the same coded moleculartag RBRB.

FIG. 5: depicts an illustrative method for detecting at least onemolecular complex or at least part of a molecular complex.

FIG. 6: schematically depicts exemplary methods and probes fordetermining the presence of nucleic acid analytes in a sample comprisingamplification.

FIG. 7: depicts exemplary coded molecular tag fabrication methods. FIG.7A schematically illustrates the fabrication of a two color codedmolecular tag using coded molecular tag subunits comprisingbacteriophage lambda genomic DNA restriction fragments, as described inExample 2. FIG. 7B depicts the generation of a coded molecular tag usingcoded molecular tag subunits comprising PCR amplicons of plasmid pBR322.

FIG. 8: depicts exemplary probe assembly methods using illustrativefabricated DNA coded molecular tags.

FIG. 9: depicts part of the metabolic pathway for the drug phenyloin inhumans. As described in Example 9, the serum levels of the analytesphenyloin, one of its active metabolites, the arene oxide of phenyloin,and a possibly toxic metabolite, 3-O-methylcatechol (shown as [PHE],[AOP], and [3OM] in FIG. 9) can be measured using the present teachings.

FIG. 10: schematically depicts an exemplary laser-confocal microscopydetection apparatus for individually detecting at least one molecularcomplex, at least one part of a molecular complex, or both, andidentifying the order of fluorescent reporter group species in at leastone identity portion, as described in Example 10.

FIG. 11: schematically depicts a substrate comprising an illustrativeelectrogenerated chemiluminescence excitation apparatus for individuallydetecting at least one bound molecular complex or at least part of amolecular complex comprising at least one electrochemiluminescentreporter group, as described in Example 11.

FIG. 12: depicts exemplary coded molecular tag fabrication methods. FIG.12A depicts fabrication of an exemplary coded molecular tag comprisingordered reporter groups using synthetic subunits. FIG. 12B depictsfluorophore-labeling an exemplary coded molecular tag comprisingaffinity tag reporter groups using appropriate fluorophore-labeledanti-affinity tag antibodies, as shown. FIG. 12C depicts an exemplarycoded molecular tag fabrication method comprising syntheticdouble-stranded coded molecular tag subunits.

FIG. 13: depicts an exemplary coded molecular tag fabrication methodusing step-wise primer extension.

DESCRIPTION OF VARIOUS EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages, regardless of the format ofsuch literature and similar materials, are expressly incorporated byreference in their entirety for any purpose.

Definitions

The term “affinity tag” as used herein refers to at least one componentof a multi-component complex, wherein the components of themulti-component complex specifically interact with or bind to eachother, for example but not limited to a capture moiety and itscorresponding capture ligand. Exemplary multiple-component complexesinclude without limitation, ligands and their receptors, including butnot limited to, avidin-biotin, streptavidin-biotin, and derivatives ofbiotin, streptavidin and/or avidin, including but not limited todesthiobiotin, NeutrAvidin, CaptAvidin, and the like; bindingproteins/peptides, including but not limited to maltose-maltose bindingprotein (MBP), calcium-calcium binding protein/peptide (CBP);antigen-antibody, including but not limited to epitope tags, includingbut not limited to c-MYC (e.g., EQKLISEEDL), HA (e.g., YPYDVPDYA), VSV-G(e.g., YTDIEMNRLGK), HSV (e.g., QPELAPEDPED), V5 (e.g., GKPIPNPLLGLDST),and FLAG Tag™ (e.g., DYKDDDDKG), and their corresponding anti-epitopeantibodies; haptens, for example but not limited to dinitrophenyl anddigoxigenin, and their corresponding antibodies; aptamers and theircorresponding targets; hybridization tags and their complements;poly-His tags (e.g., penta-His and hexa-His) and its binding partners,including without limitation, corresponding immobilized metal ionaffinity chromatography (IMAC) materials and anti-poly-His antibodies;fluorophores and anti-fluorophore antibodies; and the like. The skilledartisan will understand that at least one affinity tag can be found inone or more molecular complexes, such as in least one identity portion,at least one analytical portion, at least one reaction portion, orcombinations thereof.

The term “coded molecular tag” as used herein refers to a molecule, forexample but not limited to, a nucleic acid sequence or an amino acidsequence, comprising a multiplicity of reporter group species that areconnected, directly or indirectly to the molecule in an ordered pattern,so that the order of reporter group species can be identified when thecoded molecular tag is individually detected. In certain embodiments, atleast one coded molecular tag comprises at least two locations, referredto as labeling positions, where reporter groups are or can beincorporated, bound or attached by, but without limitation, synthesistechniques, enzymatic incorporation, chemical incorporation, reportergroup-labeled antibody binding, or binding of PNAs and/or pcPNAscomprising at least one reporter group. Typically, the occupation of atleast some labeling positions by reporter group species results in anordered pattern. This ordered pattern can be changed by adding reportergroup species to additional labeling positions or by removing orquenching reporter groups.

Typically, a coded molecular tag comprises at least one reporter groupat a particular labeling position and can comprise a multiplicity ofreporter groups at a particular labeling position. In certainembodiments, at least one coded molecular tag comprises at least onelabeling position comprising a multiplicity of reporter groups, whereinall of the reporter groups within at least one labeling position are thesame. In certain embodiments, at least one coded molecular tag comprisesat least one labeling position comprising a multiplicity of reportergroups, wherein the reporter groups within at least one labelingposition are from at least two different reporter group species. Incertain embodiments, each coded molecular tag labeling positioncomprises at least one reporter group species. In certain embodiments,at least one coded molecular tag comprises at least one labelingposition that do not comprise at least one reporter group species, i.e.,at least one of the labeling positions is vacant, but can still serve aspart of the ordered reporter group species (see, e.g., FIG. 3F, whereinthe illustrative coded molecular tag comprises the ordered pattern Y-R-Ø(i.e., vacant)-B, left to right). In certain embodiments, at least onevacant labeling position is not included in the reporter group order.

In certain embodiments, at least one coded molecular tag comprises atleast one template, for example but not limited to, at least onepeptide; at least one protein; or at least one nucleic acid sequence,such as at least part of a linear or linearizable viral genome, such asthe genomes of adenovirus, hepatitis virus, herpes virus, rotavirus, andthe like, or bacteriophages such as lambda, M13, φX-174, T-seriesbacteriophages, and the like, including derivatives thereof comprisingcloning cassettes, polylinkers, and the like; plasmids, such as pBR322and pUC series plasmids, etc., including derivatives thereof comprisingcloning cassettes, polylinkers, and the like; synthetic templates;templates comprising artificial sequences; and the like. Suitablenucleic acid templates can be double-stranded, single-stranded, or both.The skilled artisan will understand that virtually any piece of nucleicacid can serve as a template for fabricating a nucleic acid codedmolecular tag provided that it is large enough to include at least twodistinguishable labeling positions, or it can be combined with at leastone other nucleic acid sequence so that the combined sequence is largeenough to include at least two labeling positions. In certainembodiments, the restriction map and/or nucleotide sequence is known.Restriction maps and nucleotide sequences for exemplary nucleic acidtemplates can be found in, among other places, the New England BioLabs2002-03 Catalog & Technical Reference, New England BioLabs, Inc.,Beverly, Mass.; Stratagene 2003/2004 Catalog, La Jolla, Calif.; and at avariety of internet addresses, including the Entrez web site maintainedby the National Center for Biotechnology Information, particularly the“Nucleotide” web page located at world wide web address:ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide; and the BiologyWorkBench maintained by the San Diego Supercomputer Center at world wideweb address: workbench.sdsc.edu. Expressly excluded from the term codedmolecular tag is a sequence comprising a multiplicity of reporter groupsthat are not in an ordered pattern for individual detection, such asmight be used in conventional ensemble detection techniques, for examplebut not limited to, a sequence labeled with a single fluorescentreporter group species using, for example but not limited to, nicktranslation or primer extension; or a synthetic oligonucleotidecomprising incorporated reporter groups from a single reporter groupspecies.

The skilled artisan understands that the number of labeling positions ina template can vary, depending at least in part on the reporter groupspecies employed, the detection method, and sometimes the reporter groupbinding method. Generally, coded molecular tags include reporter groupspecies that are incorporated, intercalated, bound, or combinationsthereof. Typically, coded molecular tags are fabricated by combiningsubunits; hybridizing subunits on templates; synthesizing at least onesubunit on at least one template using, for example but not limited to,primer extension or PCR; binding reporter groups to templates using, forexample but not limited to, at least one reporter group-labeled PNA, atleast one reporter group-labeled pcPNA, at least one reportergroup-labeled antibody; at least one reporter group-labeled minor groovebinder; at least one reporter group-labeled aptamer; or combinationsthereof. In certain embodiments, at least one subunit is ligated to atleast one other subunit, at least one primer, or both.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, or CAB. Continuingwith this example, expressly included are combinations that containrepeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC,CBBAAA, CABABB, and so forth. The skilled artisan will understand thatthere typically is no limit on the number of items or terms in anycombination, unless otherwise apparent from the context.

The term “corresponding” as used herein refers to at least one specificrelationship between the elements to which the term refers. For example,at least one first probe of a particular probe set corresponds to atleast one second probe of the same probe set, and vice versa. The probesof a particular probe set are designed to hybridize with or bind to atleast part of a corresponding analyte, a corresponding analytesurrogate, or both; an antibody immunospecifically binds to itscorresponding antigen, or more particularly, to a corresponding epitopeof the corresponding antigen, and conversely, a particular epitope isbound by its corresponding antibody; a particular capture moiety bindsto its corresponding capture ligand, and vice versa; a particularanalyte can be identified when its corresponding molecular complex or atleast part of the corresponding molecular complex is individuallydetected and the order of the corresponding reporter group species areidentified; and so forth.

The term “diagnostic indicator” as used herein refers to at least onebiomolecule that is used as a predictor of, or is associated with, adisease state, a metabolic disorder, or the like. Exemplary diagnosticindicators include insulin; prostate specific antigen (PSA); alpha-fetalprotein (AFP); wild-type and mutant forms of cellular oncogenes andtheir protein products; wild-type and mutant forms of tumor suppressorgenes and their protein products such as p53 and pRB; rheumatoid factor;anti-nuclear antibodies; auto-antibodies; anti-foreign antigenantibodies; and the like. The skilled artisan will appreciate that forcertain diagnostic indicators, the quantitative or relative amount of,rather than the mere presence of, a particular indicator may haveclinical or biological significance. For example but without limitation,insulin levels above or below appropriate thresholds can serve as adiagnostic indicator for hyperinsulinism (hypersecretion of insulin) ordiabetes mellitus (hyposecretion of insulin); relative PSA levels orratios can serve as a diagnostic indicator for prostate cancer; relativelevels or ratios of vascular endothelial growth factor (VEGF) isoformsserve as diagnostic indicators for rheumatoid arthritis, certainmalignancies, and tumor progression; and the like. Expressly includedwithin the term diagnostic indicator are hyper- and hypo-methylatedforms of disease-related genes.

The terms “fluorophore” and “fluorescent reporter group” are intended toinclude any compound, label, or moiety that absorbs energy, typicallyfrom an illumination source, to reach an electronically excited state,and then emits energy, typically at a characteristic wavelength, toachieve a lower energy state. For example but without limitation, whencertain fluorophores are illuminated by an energy source with anappropriate excitation wavelength, typically an incandescent or laserlight source, photons in the fluorophore are emitted at a characteristicfluorescent emission wavelength. Fluorophores, sometimes referred to asfluorescent dyes, may typically be divided into families, such asfluorescein and its derivatives; rhodamine and its derivatives; cyanineand its derivatives; coumarin and its derivatives; Cascade Blue™ and itsderivatives; Lucifer Yellow and its derivatives; BODIPY and itsderivatives; and the like. Exemplary fluorophores includeindocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5,Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa Fluor 488, AlexaFluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, AlexaFluor 647, Alexa Fluor 660, Alexa Fluor 680, JOE, Lissamine, RhodamineGreen, BODIPY, fluorescein isothiocyanate (FITC), carboxy-fluorescein(FAM), phycoerythrin, rhodamine, dichlororhodamine (dRhodamine™),carboxy tetramethylrhodamine (TAMRA™), carboxy-X-rhodamine (ROX™), LIZ™,VIC™, NED™, PET™, SYBR, PicoGreen, RiboGreen, and the like. Descriptionsof fluorophores and their use, can be found in, among other places, R.Haugland, Handbook of Fluorescent Probes and Research Products, 9^(th)ed. (2002), Molecular Probes, Eugene, Oreg.; M. Schena, MicroarrayAnalysis (2003), John Wiley & Sons, Hoboken, N.J.; Synthetic MedicinalChemistry 2003/2004 Catalog, Berry and Associates, Ann Arbor, Mich.; G.Hermanson, Bioconjugate Techniques, Academic Press (1996); and GlenResearch 2002 Catalog, Sterling, Va. Near-infrared dyes are expresslywithin the intended meaning of the terms fluorophore and fluorescentreporter group.

The term “foreign antigen” as used herein refers to one or morecomponents of, metabolic products of, or one or more element derivedfrom a foreign organism. Exemplary foreign organisms include bacteria,fungi, protozoa, viruses, insects, parasites, and other infectiousand/or pathogenic agents. A foreign antigen typically comprises at leastone protein, including but not limited to glycoproteins,phosphoproteins, lipoproteins, flagellin, peptidoglycan, endotoxin, andexotoxin; at least one peptide; at least one lipopolysaccharide; atleast one prion; at least one nucleic acid; and the like.

The term “hybridization tag” as used herein refers to an oligonucleotidesequence that can be used for separating the element to which it isbound, including without limitation, bulk separation; or tethering orattaching a multiplicity of hybrid pairs comprising different elementspecies and the same hybridization tag species to a substrate, or both.In certain embodiments, the same hybridization tag is used with amultiplicity of different elements to effect: bulk separation, substratetethering, substrate attachment, or combinations thereof. Ahybridization tag complement typically refers to at least oneoligonucleotide that comprises at least one sequence of nucleotides thatare complementary to and hybridize with the hybridization tag. Invarious embodiments, hybridization tag complements serve as capturemoieties for tethering or attaching at least one hybridizationtag:element complex to at least one substrate; serve as “pull-out”sequences for bulk separation procedures; or both as capture moietiesand as pull-out sequences.

Typically, hybridization tags and their corresponding hybridization tagcomplements are selected to minimize: internal, self-hybridization;cross-hybridization with different hybridization tag species, nucleotidesequences in a sample, including but not limited to analytes,hybridization tag complements, or analyte surrogates; but should beamenable to facile hybridization between the hybridization tag and itscorresponding hybridization tag complement. Hybridization tag sequencesand hybridization tag complement sequences can be selected by anysuitable method, for example but not limited to, computer algorithmssuch as described in PCT Publication Nos. WO 96/12014 and WO 96/41011and in European Publication No. EP 799,897; and the algorithm andparameters of SantaLucia (Proc. Natl. Acad. Sci. 95:1460-65 (1998)).Descriptions of hybridization tags can be found in, among other places,U.S. Pat. No. 6,309,829 (referred to as “tag segment” therein); U.S.Pat. No. 6,451,525 (referred to as “tag segment” therein); U.S. Pat. No.6,309,829 (referred to as “tag segment” therein); U.S. Pat. No.5,981,176 (referred to as “grid oligonucleotides” therein); U.S. Pat.No. 5,935,793 (referred to as “identifier tags” therein); and PCTPublication No. WO 01/92579 (referred to as “addressablesupport-specific sequences” therein).

Hybridization tags can be attached to at least one end of at least oneprobe; or they can be located internally, for example but not limitedto, adjacent to at least one restriction enzyme cleavage site, adjacentto at least one cleavable crosslinker, or both, such that cleavage atthe restriction enzyme site or the cleavable crosslinker will result inthe hybridization tag being at or near the newly-created end. In certainembodiments, at least one hybridization tag comprises or overlaps atleast one restriction enzyme cleavage site. In certain embodiments,hybridization tags are at least 12 bases in length, at least 15 bases inlength, 12-60 bases in length, or 15-30 bases in length. In certainembodiments, at least one hybridization tag is 12, 15, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 45, or 60 bases in length. In certainembodiments, at least two hybridization tag:hybridization tag complementduplexes have melting temperatures that fall within a ΔTm range(Tmax−Tmin) of no more than 10° C. of each other. In certainembodiments, at least two hybridization tag:hybridization tag complementduplexes have melting temperatures that fall within a ΔTm range of 5° C.or less of each other.

In certain embodiments, at least one hybridization tag is used toseparate the element to which it is bound from at least one unboundcomponent in a sample, unbound components and/or reagents in thereaction mixture, or the like. In certain embodiments, hybridizationtags are used to attach at least one molecular complex or at least partof at least one molecular complex to at least one substrate. In certainembodiments, a multiplicity of molecular complexes, a multiplicity ofcleavable components, a multiplicity of identity portions, amultiplicity of coded molecular tags, or combinations thereof, comprisethe same hybridization tag. For example but not limited to, separating amultiplicity of different element:hybridization tag species using thesame hybridization tag complement, tethering a multiplicity of differentelement:hybridization tag species to a substrate comprising the samehybridization tag complement, or both.

The term “individually detecting” as used herein refers to the processof evaluating and/or interrogating the reporter group species ofseparate, discrete molecular complexes or at least parts of molecularcomplexes, in contrast to ensemble detection of reporter group speciesin populations of molecular complexes, as routinely done, for example,in microarray or immunoassay techniques. Typically, the order ofreporter group species in at least one individually detected molecularcomplex or at least part of a molecular complex is determined, relativeto a reference or orientation point, for example but not limited to, atethering site, attachment sites, or both; or a set of coded moleculartags in which one or more particular labeling sites are always occupiedby the same reporter group species, i.e., a distinguishable sub-pattern.Expressly excluded from the term individually detecting are techniquesthat comprise cleaving or releasing multiple subunits from a polymer anddetecting the reporter groups of such cleaved subunits in a piecemealfashion to determine their position or sequence in the intact polymer,such as nucleic acid sequencing or restriction enzyme mappingtechniques.

The term “mobility modifier” as used herein refers to at least onemolecular entity, for example but not limited to, at least one polymerchain, that when added to at least one element (e.g., at least oneprobe, at least one identity portion, at least one coded molecular tag,at least one molecular complex, at least one cleavable component, orcombinations thereof) affects the mobility of the element to which it ishybridized or bound, covalently or non-covalently, in at least onemobility-dependent analytical technique. In certain embodiments, amultiplicity of probe sets exclusive of mobility modifiers, amultiplicity of molecular complexes exclusive of mobility modifiers, amultiplicity of identity portions exclusive of mobility modifiers, amultiplicity of cleavable components exclusive of mobility modifiers, amultiplicity of coded molecular tags exclusive of mobility modifiers, orcombinations thereof, have the same or substantially the same mobilityin at least one mobility-dependent analytical technique. Typically, amobility modifier changes the charge/translational frictional drag whenhybridized or bound to the element; or imparts a distinctive mobility,for example but not limited to, a distinctive elution characteristic ina chromatographic separation medium or a distinctive electrophoreticmobility in a sieving matrix or non-sieving matrix, when hybridized orbound to the corresponding element; or both (see, e.g., U.S. Pat. Nos.5,470,705 and 5,514,543).

A mobility-dependent analytical technique is a technique based ondifferential rates of migration between different species beingseparated. Exemplary mobility-dependent analysis techniques includeelectrophoresis, chromatography, mass spectroscopy, sedimentation, e.g.,gradient centrifugation, field-flow fractionation, multi-stageextraction techniques and the like. Descriptions of mobility-dependentanalytical techniques can be found in, among other places, U.S. Pat.Nos. 5,470,705, 5,514,543, 5,580,732, 5,624,800, and 5,807,682 and PCTPublication No. WO 01/92579.

In certain embodiments, a multiplicity of molecular complexes comprisingmobility modifiers, a multiplicity of cleavable components comprisingmobility modifiers, a multiplicity of identity portions comprisingmobility modifiers, a multiplicity of coded molecular tags comprisingmobility modifiers, or combinations thereof, have substantially similardistinctive mobilities, for example but not limited to, when amultiplicity of elements comprising mobility modifiers havesubstantially similar distinctive mobilities so they can be bulkseparated or they can be separated from other elements comprisingmobility modifiers with different distinctive mobilities. In certainembodiments, a multiplicity of molecular complexes comprising mobilitymodifiers, a multiplicity of cleavable components comprising mobilitymodifiers, a multiplicity of identity portions comprising mobilitymodifiers, a multiplicity of coded molecular tags comprising mobilitymodifiers, or combinations thereof, have different distinctivemobilities.

In certain embodiments, at least one mobility modifier comprises atleast one nucleotide polymer chain, including without limitation, atleast one oligonucleotide polymer chain, at least one polynucleotidepolymer chain, or both at least one oligonucleotide polymer chain and atleast one polynucleotide polymer chain. In certain embodiments, at leastone mobility modifier comprises at least one non-nucleotide polymerchain. Exemplary non-nucleotide polymer chains include, withoutlimitation, peptides, polypeptides, polyethylene oxide (PEO), or thelike. In certain embodiments, at least one polymer chain comprises atleast one substantially uncharged, water-soluble chain, such as a chaincomposed of PEO units; a polypeptide chain; or combinations thereof.

The polymer chain can comprise a homopolymer, a random copolymer, ablock copolymer, or combinations thereof. Furthermore, the polymer chaincan have a linear architecture, a comb architecture, a branchedarchitecture, a dendritic architecture (e.g., polymers containingpolyamidoamine branched polymers, Polysciences, Inc. Warrington, Pa.),or combinations thereof. In certain embodiments, at least one polymerchain is hydrophilic, or at least sufficiently hydrophilic whenhybridized or bound to an element to ensure that the element-mobilitymodifier is readily soluble in aqueous medium. Where themobility-dependent analysis technique is electrophoresis, in certainembodiments, the polymer chains are uncharged or have a charge/subunitdensity that is substantially less than that of its correspondingelement.

The synthesis of polymer chains useful as mobility modifiers willdepend, at least in part, on the nature of the polymer. Methods forpreparing suitable polymers generally follow well-known polymer subunitsynthesis methods. These methods, which involve coupling ofdefined-size, multi-subunit polymer units to one another, eitherdirectly or through charged or uncharged linking groups, are generallyapplicable to a wide variety of polymers, such as polyethylene oxide,polyglycolic acid, polylactic acid, polyurethane polymers, polypeptides,oligosaccharides, and nucleotide polymers. Such methods of polymer unitcoupling are also suitable for synthesizing selected-length copolymers,e.g., copolymers of polyethylene oxide units alternating withpolypropylene units. Polypeptides of selected lengths and amino acidcomposition, either homopolymer or mixed polymer, can be synthesized bystandard solid-phase methods (e.g., Int. J. Peptide Protein Res., 35:161-214 (1990)).

One method for preparing PEO polymer chains having a selected number ofhexaethylene oxide (HEO) units, an HEO unit is protected at one end withdimethoxytrityl (DMT), and activated at its other end with methanesulfonate. The activated HEO is then reacted with a second DMT-protectedHEO group to form a DMT-protected HEO dimer. This unit-addition is thencarried out successively until a desired PEO chain length is achieved(e.g., U.S. Pat. No. 4,914,210; see also, U.S. Pat. No. 5,777,096).

The term “molecular complex” as used herein refers to a reactionproduct, comprising at least one identity portion comprising at leastone coded molecular tag, formed due to the presence of a particularanalyte in the sample. By individually detecting a particular molecularcomplex or at least a part of that molecular complex, one can determinethat the corresponding analyte is present in the sample. The molecularcomplex may, but need not, comprise all or part of the correspondinganalyte or analyte surrogate, as shown for example, in FIG. 1A. Incertain embodiments, at least one molecular complex comprises at leastone analyte or at least one analyte surrogate and at least one probecomprising at least one identity portion. In certain embodiments, one ormore molecular complexes comprise a single “linked” molecule, forexample but not limited to, a ligation product molecular complex, shownas MC1 and MC2 in FIG. 1A. The skilled artisan will understand thatligation product molecular complexes are a subset of the term molecularcomplex, as are analytes and/or analyte surrogates hybridized with atleast one ligation product molecular complex. In certain embodiments, atleast one molecular complex comprises an assembly comprising at leasttwo interacting or bound molecules, for example but not limited to anantigen-antibody complex, an aptamer-target complex, anantibody-antigen-aptamer complex, or the like, for example, as shown inFIG. 1A (e.g., 2:1P2:2P2B), FIG. 1B (MC1 and MC2), and FIG. 6B (MC1 andMC2).

In certain embodiments, at least one molecular complex comprises atleast one analyte surrogate and at least one probe comprising at leastone identity portion. An analyte surrogate typically comprises anamplification product, such as a cDNA, an amplicon, a primer extensionproduct, a transcription product, a translation product, an LCR product,or the like, that results from amplifying at least part of at least oneanalyte or at least part of at least one analyte surrogate, buttypically does not comprise the original analyte. Expressly excludedfrom the term molecular complex are entities or assemblies comprisingone or more bead or particle, such as latex beads, agarose beads,magnetic and paramagnetic particles, dye-impregnated polymer beads,metallic particles, and the like.

In certain embodiments, at least one analyte comprises at least oneamino acid, at least one nucleotide, at least one oligosaccharide, atleast one phosphodiester linkage, at least one peptide bond, at leastone glycosidic bond, or combinations thereof. In certain embodiments, atleast one analyte comprises at least one biomolecule; at least one drug;at least one small molecule for example but not limited to a smallorganic molecule or metabolite; or combinations thereof. In certainembodiments, at least one analyte comprises at least one polynucleotide,such as at least one nucleic acid sequence, including but not limited toat least one genomic DNA (gDNA); hnRNA; mRNA; noncoding RNA (ncRNA),including but not limited to rRNA, tRNA, mRNA (micro RNA), siRNA (smallinterfering RNA), snoRNA (small nucleolar RNA), snRNA (small nuclearRNA) and stRNA (small temporal RNA); fragmented nucleic acid; nucleicacid obtained from subcellular organelles such as mitochondria orchloroplasts; and nucleic acid obtained from microorganisms, parasites,or DNA or RNA viruses that may be present in a sample. Furthermore, anucleic acid analyte can be present in double-stranded form,single-stranded form, or both double-stranded and single-stranded form.Discussions of nucleic acid analytes can be found in, among otherplaces, Current Protocols in Nucleic Acid Chemistry, S. Beaucage, D.Bergstrom, G. Glick, and R. Jones, eds., John Wiley & Sons (1999)including updates through August 2003.; S. Verma and F. Eckstein, Ann.Rev. Biochem., 67:99-134 (1998); S. Buckingham, Horizon Symposia,Understanding the RNAissance, Nature Publishing Group, May 2003 at pages1-3; S. Eddy, Nature Rev. Genetics 2:919-29 (2001); and Nucleic Acids inChemistry and Biology, 2d ed., G. Blackburn and M. Gait, eds., OxfordUniversity Press (1996). In certain embodiments, the compositions,methods, and kits disclosed herein, can be used to analyze heritableand/or somatic mutations, including but not limited to nonsensemutations, missense mutations, insertions, deletions, and chromosomaltranslocations at the DNA, RNA, or protein levels.

In certain embodiments, at least one analyte comprises at least onepeptide bond such as found in peptides, oligopeptides, and proteins. Incertain embodiments, at least one analyte comprises at least one foreignantigen. In certain embodiments, at least one analyte comprises at leastone diagnostic indicator. In certain embodiments, at least one analytecomprises at least one antibody molecule or at least one fragment orcomponent of an antibody molecule. In certain embodiments, at least oneanalyte comprises at least one glycosidic bond, such as found indisaccharides, oligosaccharides, and polysaccharides, including but notlimited to sugar residues present in glycoproteins.

The person of ordinary skill will appreciate that while the targetsequence of a nucleic acid analyte or analyte surrogate can be describedas a single-stranded molecule, the opposing strand of a double-strandedanalyte comprises a complementary sequence that can also be used as atarget for probe hybridization. In certain embodiments, a targetsequence comprises an upstream or 5′ region, a downstream or 3′ region,and a “pivotal nucleotide” located at the junction of the upstreamregion and the downstream region (e.g., shown as “X” in FIG. 2; seealso, PCT Publication No. WO 01/92579). In certain embodiments, thepresence or absence of the pivotal nucleotide is being detected by theprobe set and may represent, for example, without limitation, a singlepolymorphic nucleotide in a multi-allelic target locus, a heritable orsomatic mutation, or the like.

FIG. 3 schematically depicts exemplary molecular complexes. In eachpanel, the identity portion is illustrated as an open (unfilled)rectangle, the reaction portions are illustrated as a dotted rectangle,the analytical portion is illustrated as a diagonally striped rectangle;and the reporter groups are designated R, G, B, and Y, for example butnot limited to, four different fluorescent reporter group species. Inpanel A, the exemplary molecular complex includes an identity portioncomprising individual reporter groups R, G, B, and Y, in that order(throughout this disclosure, the order of reporter group species isshown L to R for illustration purposes, unless otherwise apparent fromthe context); and an analytical portion comprising at least one biotinmoiety (b). The exemplary molecular complex depicted in panel B includesan identity portion comprising individual reporter groups R, G, B, andY, in that order; and an analytical portion comprising at least oneepitope tag (ET). Panel C depicts another exemplary molecular complexcomprising an analytical portion comprising at least one mobilitymodifier (MM); a reaction portion comprising at least one biotin moiety(b); and an identity portion comprising reporter groups Y, B, and R, inthe order YBBR. The exemplary molecular complex shown in panel Dincludes an identity portion comprising reporter group species R, G, B,and Y, in that order (i.e., in labeling positions 4, 3, 2, and 1,respectively), wherein each occupied coded molecular tag labelingposition comprises at least one reporter group and in some cases amultiplicity of reporter groups, typically the same reporter groupspecies but possibly more than one reporter group species, for exampleto provide color complementation at that labeling site; and ananalytical portion comprising at least one hybridization tag (“HT”). Inpanel E, another exemplary molecular complex is shown, comprising ananalytical portion comprising at least one aptamer sequence (“apt”); andan identity portion comprising a coded molecular tag including fourspaced reporter group species, B, G, R, and Y in labeling positions 4,3, 2, and 1, respectively. The exemplary molecular complex shown inpanel F includes an analytical portion comprising at least one epitopetag (ET); a cleavable linker (dark region); and an identity portioncomprising at least one biotin moiety and three reporter group species,Y, R, and B, in labeling positions 1, 2, and 4, respectively. Noreporter groups are present at labeling position 3 (shown as Ø). Thus,when individually detected, the order of reporter groups in thecleavable identity portion is Y, R, blank (empty), and B, relative tothe illustrated biotin moiety.

The term “polymerase” is used in a broad sense herein and includes DNApolymerases, enzymes that typically synthesize DNA by incorporatingdeoxyribonucleotide triphosphates or analogs in the 5′=>3′ direction ina template-dependent and primer-dependent manner; RNA polymerases,enzymes that synthesize RNA by incorporating ribonucleotidetriphosphates or analogs and may or may not be in a template-dependentmanner; and reverse transcriptases, also known as RNA-dependent DNApolymerases, that synthesize DNA by incorporating deoxyribonucleotidetriphosphates or analogs in the 5′=>3′ direction in primer-dependentmanner, typically using an RNA template. Descriptions of polymerases canbe found in, among other places, R. M. Twyman, Advanced MolecularBiology, Bios Scientific Publishers Ltd. (1999); Polymerase EnzymeResource Guide, Promega, Madison, Wis. (1998); P. C. Turner et al.,Instant Notes in Molecular Biology, Bios Scientific Publishers Ltd.(1997); and B. D. Hames et al., Instant Notes in Biochemistry, BiosScientific Publishers Ltd. (1997).

The term “polynucleotide” means polymers comprising at least twonucleotide monomers, including analogs of such polymers, includingdouble and single stranded deoxyribonucleotides, ribonucleotides,α-anomeric forms thereof, and the like. Monomers are linked by“internucleotide linkages,” e.g., phosphodiester linkages, where as usedherein, the term “phosphodiester linkage” refers to phosphodiester bondsor bonds including phosphate analogs thereof, including associatedcounterions, e.g., H⁺, NH₄ ⁺, Na⁺, if such counterions are present.Whenever a DNA polynucleotide is represented by a sequence of letters,such as “ATGCCTG,” it will be understood that the nucleotides are in 5′to 3′ order from left to right, unless it is otherwise apparent from thecontext, and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine,“G” denotes deoxyguanosine, and “T” denotes deoxythymidine, unlessotherwise noted.

“Analogs”, in reference to nucleosides and/or polynucleotides, comprisesynthetic analogs having modified nucleobase portions, modified pentoseportions and/or modified phosphate portions, and, in the case ofpolynucleotides, modified internucleotide linkages, as describedgenerally elsewhere (e.g., Scheit, Nucleotide Analogs (John Wiley, NewYork, (1980); Englisch, Angew. Chem. Int. Ed. Engl. 30:613-29 (1991);Agarwal, Protocols for Polynucleotides and Analogs, Humana Press (1994);and S. Verma and F. Eckstein, Ann. Rev. Biochem. 67:99-134 (1999)).Generally, modified phosphate portions comprise analogs of phosphatewherein the phosphorous atom is in the +5 oxidation state and one ormore of the oxygen atoms is replaced with a non-oxygen moiety, e.g.,sulfur. Exemplary phosphate analogs include but are not limited tophosphorothioate, phosphorodithioate, methylphosphonates,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, boronophosphates, includingassociated counterions, e.g., H⁺, NH₄ ⁺, Na⁺, if such counterions arepresent. Exemplary modified nucleobase portions include but are notlimited to 2,6-diaminopurine, hypoxanthine, pseudouridine, C-5-propyne,isocytosine, isoguanine, 2-thiopyrimidine, and other like analogs.Particularly preferred nucleobase analogs are iso-C and iso-G nucleobaseanalogs available from Sulfonics, Inc., Alachua, Fla. (e.g., Benner, etal., U.S. Pat. No. 5,432,272) or LNA analogs (e.g., Koshkin et al.,Tetrahedron 54:3607-30 (1998)). Exemplary modified pentose portionsinclude but are not limited to 2′- or 3′-modifications where the 2′- or3′-position is hydrogen, hydroxy, alkoxy, e.g., methoxy, ethoxy,allyloxy, isopropoxy, butoxy, isobutoxy and phenoxy, azido, amino oralkylamino, fluoro, chloro, bromo and the like. Modified internucleotidelinkages include phosphate analogs, analogs having achiral and unchargedintersubunit linkages (e.g., E. Sterchak et al., Organic Chem., 52:4202(1987)), and uncharged morpholino-based polymers having achiralintersubunit linkages (e.g., U.S. Pat. No. 5,034,506). Preferredinternucleotide linkage analogs include PNA, pcPNA, morpholidate,acetal, and polyamide-linked heterocycles. A particularly preferredclass of polynucleotide analogs where a conventional sugar andinternucleotide linkage has been replaced with a 2-aminoethylglycineamide backbone polymer is PNA and pcPNA (e.g., Nielsen et al., Science,254:1497-1500 (1991); Egholm et al., J. Am. Chem. Soc., 114: 1895-1897(1992)). Detailed descriptions of oligonucleotide synthesis and analogs,including relevant protocols can be found in, among other places, S.Verma and F. Eckstein, Ann. Rev. Biochem. 67:99-134 (1999); J.Goodchild, Bioconj. Chem. 1:165-87 (1990); S. L. Beaucage et al.,Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, NewYork, N.Y. (2000); U.S. Pat. Nos. 4,373,071; 4,401,796; 4,415,732;4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,047,524; 5,132,418;5,153,319; and 5,262,530.

The term “reporter group” is used in a broad sense herein and refers toany identifiable tag, label, or moiety. The skilled artisan willappreciate that many different species of reporter groups can be used inthe present teachings, either individually or in combination with one ormore different reporter group. Exemplary reporter groups include, butare not limited to, fluorophores, radioisotopes, chromogens, enzymes,antigens including but not limited to epitope tags, heavy metals, dyes,phosphorescence groups, chemiluminescent groups, electrochemicaldetection moieties, affinity tags, binding proteins, phosphors, rareearth chelates, near-infrared dyes, including but not limited to,“Cy.7.5Ph.NCS,” “Cy.7.OphEt.NCS,” “Cy7.OphEt.CO₂Su”, and IRD800 (see,e.g., J. Flanagan et al., Bioconjug. Chem. 8:751-56 (1997); and DNASynthesis with IRD800 Phosphoramidite, LI-COR Bulletin #111, LI-COR,Inc., Lincoln, Nebr.), electrochemiluminescence labels, including butnot limited to, tris(bipyridal) ruthenium (II), also known as Ru(bpy)₃²⁺, Os(1,1-phenanthroline)₂bis(diphenylphosphino)ethane²⁺, also known asOs(phen)₂(dppene)²⁺, luminol/hydrogen peroxide,Al(hydroxyquinoline-5-sulfonic acid),9,10-diphenylanthracene-2-sulfonate, andtris(4-vinyl-4′-methyl-2,2′-bipyridal) ruthenium (II), also known asRu(v-bpy₃ ²⁺), and the like.

The term reporter group also includes at least one element ofmulti-element indirect reporter systems, e.g., affinity tags such asbiotin/avidin, antibody/antigen, ligand/receptor including but notlimited to binding proteins and their ligands, enzyme/substrate, and thelike, in which one element interacts with other elements of the systemin order to effect the potential for a detectable signal. Exemplarymulti-element reporter system include a probe comprising at least onebiotin reporter group with an streptavidin-conjugated fluorophore, orvice versa; a probe comprising at least one dinitrophenyl (DNP) reportergroup and a fluorophore-labeled anti-DNP antibody; and the like.Detailed protocols for methods of attaching reporter groups tooligonucleotides, polynucleotides, peptides, proteins, mono-, di- andoligosaccharides, organic molecules, and the like can be found in, amongother places, G. T. Hermanson, Bioconjugate Techniques, Academic Press,San Diego, Calif. (1996)(“Bioconjugate Techniques”); S. L. Beaucage etal., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, NewYork, N.Y. (2000); Handbook of Fluorescent Probes and Research Products,9^(th) ed., Molecular Probes, Inc., Eugene, Oreg. (2002); and PierceApplications Handbook and Catalog 2003-2004, Pierce Biotechnology,Rockford, Ill. (2003).

In certain embodiments, at least one reporter group comprises anelectrochemiluminescent moiety that can, under appropriate conditions,emit detectable electrogenerated chemiluminescence (ECL). In ECL,excitation of the electrochemiluminescent moiety is electrochemicallydriven and the chemiluminescent emission can be optically detected.Exemplary electrochemiluminescent reporter group species include:Ru(bpy)₃ ²⁺ and Ru(v-bpy)₃ ²⁺ with emission wavelengths of 620 nm;Os(phen)₂(dppene)²⁺ with an emission wavelength of 584 nm;luminol/hydrogen peroxide with an emission wavelength of 425 nm;Al(hydroxyquinoline-5-sulfonic acid) with an emission wavelength of 499nm; and 9,10-diphenylanothracene-2-sulfonate with an emission wavelengthof 428 nm; and the like. Modified forms of these threeelectrochemiluminescent reporter group species that are amenable toincorporation into probes and coded molecular tags are commerciallyavailable or can be synthesized without undue experimentation usingtechniques known in the art. For example, there is a Ru(bpy)₃ ²⁺N-hydroxy succinimide ester for coupling to nucleic acid sequencesthrough an amino linker group has been described (see, U.S. Pat. No.6,048,687); and succinimide esters of Os(phen)₂(dppene)²⁺ and Al(HQS)₃³⁺ can be synthesized and attached to nucleic acid sequences usingsimilar methods. The Ru(bpy)₃ ²⁺ electrochemiluminescent reporter groupcan be synthetically incorporated into nucleic acid sequences usingcommercially available ru-phosphoramidite (IGEN International, Inc.,Gaithersburg, Md.).

Additionally other polyaromatic compounds and chelates of ruthenium,osmium, platinum, palladium, and other transition metals have shownelectrochemiluminescent properties. Detailed descriptions of ECL andelectrochemiluminescent moieties can be found in, among other places, A.Bard and L. Faulkner, Electrochemical Methods, John Wiley & Sons (2001);M. Collinson and M. Wightman, Anal. Chem. 65:2576 et seq. (1993); D.Brunce and M. Richter, Anal. Chem. 74:3157 et seq. (2002); A. Knight,Trends in Anal. Chem. 18:47 et seq. (1999); B. Muegge et al., Anal.Chem. 75:1102 et seq. (2003); H. Abrunda et al., J. Amer. Chem. Soc.104:2641 et seq. (1982); K. Maness et al., J. Amer. Chem. Soc. 118:10609et seq. (1996); M. Collinson and R. Wightman, Science 268:1883 et seq.(1995); and U.S. Pat. No. 6,479,233.

The term “sample” is used in a broad sense herein and is intended toinclude a wide range of biological materials as well as compositionsderived or extracted from such biological materials. Exemplary samplesinclude whole blood; red blood cells; white blood cells; buffy coat;hair; nails and cuticle material; swabs, including but not limited tobuccal swabs, throat swabs, vaginal swabs, urethral swabs, cervicalswabs, throat swabs, rectal swabs, lesion swabs, abcess swabs,nasopharyngeal swabs, and the like; urine; sputum; saliva; semen;lymphatic fluid; amniotic fluid; cerebrospinal fluid; peritonealeffusions; pleural effusions; fluid from cysts; synovial fluid; vitreoushumor; aqueous humor; bursa fluid; eye washes; eye aspirates; plasma;serum; pulmonary lavage; lung aspirates; and tissues, including but notlimited to, liver, spleen, kidney, lung, intestine, brain, heart,muscle, pancreas, biopsy material, and the like. The skilled artisanwill appreciate that lysates, extracts, or material obtained from any ofthe above exemplary biological samples are also within the scope of theinvention. Tissue culture cells, including explanted material, primarycells, secondary cell lines, and the like, as well as lysates, extracts,or materials obtained from any cells, are also within the meaning of theterm biological sample as used herein. Microorganisms and viruses thatmay be present on or in a sample are also within the scope of theinvention. Materials obtained from forensic settings are also within theintended meaning of the term sample.

The term “substrate” as used herein refers to one or more surfaces thata molecular complex or at least part of a molecular complex can interactwith or bind to, either directly or indirectly. Substrate surfaces aretypically planar, but can comprise a wide variety of topographies,including combinations of topographies on the same surface. The skilledartisan will appreciate that the suitability of a particular substrate,including its topography and composition, typically depends on thetype(s) of molecular complex to be detected and the detectiontechnique(s) employed.

II. Reagents

As used herein, the terms antibody and antibodies are used in a broadsense, to include not only intact antibody molecules, for example butnot limited to immunoglobulin A, immunoglobulin G and immunoglobulin M,but also any immunoreactive component(s) of an antibody molecule thatimmunospecifically bind to at least one epitope. Such immunoreactivecomponents include but are not limited to, FAb fragments, FAb′fragments, FAb′2 fragments, single chain antibody fragments (scFv),miniantibodies, diabodies, crosslinked antibody fragments, Affibody®molecules, and the like. Immunoreactive products derived using antibodyengineering or protein engineering techniques are also expressly withinthe meaning of the term antibodies. Detailed descriptions of antibodyand/or protein engineering, including relevant protocols, can be foundin, among other places, J. Maynard and G. Georgiou, Ann. Rev. Biomed.Eng. 2:339-76 (2000); Antibody Engineering, R. Kontermann and S. Dübel,eds., Springer Lab Manual, Springer Verlag (2001); A. Worn and A.Pluckthun, J. Mol. Biol. 305:989-1010 (2001); J. McCafferty et al.,Nature 348:552-54 (1990); Müller et al., FEBS Letter, 432:45-9 (1998);A. Plückthun and P. Pack, Immunotechnology, 3:83-105 (1997); U.S. Pat.No. 5,831,012; and S. Paul, Antibody Engineering Protocols, Humana Press(1995).

The skilled artisan will appreciate that antibody can be obtained from avariety of sources, including but not limited to polyclonal antibody,monoclonal antibody, monospecific antibody, recombinantly expressedantibody, humanized antibody, plantibodies, and the like; and can beobtained from a variety of animal species, including rabbit, mouse,goat, rat, human, horse, bovine, guinea pig, chicken, sheep, donkey,human, and the like. A wide variety of antibody is commerciallyavailable and custom-made antibody can be obtained from a number ofcontract labs. Detailed descriptions of antibodies, including relevantprotocols, can be found in, among other places, Current Protocols inImmunology, Coligan et al., eds., John Wiley & Sons (1999, includingupdates through August 2003); The Electronic Notebook; Basic Methods inAntibody Production and Characterization, G. Howard and D. Bethel, eds.,CRC Press (2000); J. Goding, Monoclonal Antibodies: Principles andPractice, 3d Ed., Academic Press (1996); E. Harlow and D. Lane, UsingAntibodies, Cold Spring Harbor Lab Press (1999); P. Shepherd and C.Dean, Monoclonal Antibodies: A Practical Approach, Oxford UniversityPress (2000); A. Johnstone and M. Turner, Immunochemistry 1 and 2,Oxford University Press (1997); C. Borrebaeck, Antibody Engineering, 2ded., Oxford university Press (1995); A. Johnstone and R. Thorpe,Immunochemistry in Practice, Blackwell Science, Ltd. (1996); H. Zola,Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies andEngineered Antibody Derivatives (Basics: From Background to Bench),Springer Verlag (2000); and S. Hockfield et al., Selected Methods forAntibody and Nucleic Acid Probes, Cold Spring Harbor Lab Press (1993).Additionally, a vast number of commercially available antibodies,including labeled or unlabeled; polyclonal, monoclonal, and monospecificantibodies, as well as immunoreactive components thereof; customantibody suppliers, and the like can be found on the World Wide Web at,among other places, the Antibody Search page at biocompare.com, theAntibody Resource Page at antibodyresource.com, and the AntibodyExplorer page at sigmaaldrich.com.

Aptamers include nucleic acid aptamers (i.e., single-stranded DNAmolecules or single-stranded RNA molecules) and peptide aptamers.Aptamers bind target molecules in a highly specific,conformation-dependent manner, typically with very high affinity,although aptamers with lower binding affinity can be selected ifdesired. Aptamers have been shown to distinguish between targets basedon very small structural differences such as the presence or absence ofa methyl or hydroxyl group and certain aptamers can distinguish betweenD- and L-enantiomers. Aptamers have been obtained that bind smallmolecular targets, including drugs, metal ions, and organic dyes,peptides, biotin, and proteins, including but not limited tostreptavidin, VEGF, and viral proteins. Aptamers have been shown toretain functional activity after biotinylation, fluorescein labeling,and when attached to glass surfaces and microspheres.

Nucleic acid aptamers, including speigelmers, are identified by an invitro selection process known as systematic evolution of ligands byexponential amplification (SELEX). In the SELEX process very largecombinatorial libraries of oligonucleotides, for example 10¹⁴ to 10¹⁵individual sequences, often as large as 60-100 nucleotides long, areroutinely screened by an iterative process of in vitro selection andamplification. Most targets are affinity enriched within 8-15 cycles andthe process has been automated allowing for faster aptamer isolation.Peptide aptamers are typically identified by several different proteinengineering techniques known in the art, including but not limited to,phage display, ribosome display, mRNA display, selectively infectedphage technology (SIP), and the like. The skilled artisan willunderstand that nucleic acid aptamers and peptide aptamers can beobtained following conventional procedures and without undueexperimentation. Detailed descriptions of aptamers, including relevantprotocols, can be found in, among other places, L. Gold, J. Biol. Chem.,270(23):13581-84 (1995); L. Gold et al., Ann. Rev. Biochem. 64:763-97(1995); S. Jayashena, Clin. Chem., 45:1628-50 (1999); V. Sieber et al.,Nat. Biotech. 16:955-60 (1998); L. Jermutus et al., Curr. Opin. Biotech.9:534-48 (1998); D. Wilson and J. Szostak, Ann. Rev. Biochem. 68:611-47(1999); L. Jermutus et al., Eur. Biophys. J., 31:179-84 (2002); G.Connell et al., Biochem., 32:5497-5502 (1993); M. Famulok et al., Acc.Chem. Res. 33:591-99 (2000); W. James, Curr. Opin. Pharmacol., 1:540-46(2001); J. Cox. Et al., Nucl. Acid Res. 30(20):e18 (2002); S. Clark andV. Remcho, Electrophoresis 23:1335-40, 2002; A. Tahiri-Alaoui et al.,Nuc. Acid Res. 30(10):e45 (2002); A. Kopylov and V. Spiridonova,Molecular Biology 34:940-54 (2000); J. Blum et al., Proc. Natl. Acad.Sci., 97:2241-46 (2000); Phage Display: A Laboratory Manual, C. Barbas,D. Burton, J. Scott, and G. Silverman, eds., Cold Spring HarborLaboratory Press (2001); S. Jung et al., J. Mol. Biol. 294:163-80(1999); N. Raffler et al., Chem. & Biol., 10:69-79 (2003); A. Plückthunet al., Adv. Protein Chem. 55:367-403 (2000); Amstutz et al., Curr.Opin. Biotech., 12:400-05 (2001); J. Hanes and A. Pluckthun, Proc. Natl.Acad. Sci., 94:4937-42 (1997); Protein-Protein Interactions, A MolecularCloning Manual, E. Golemis, ed., Cold Spring Harbor Press (2001); C.Krebber et al., J. Mol. Biol. 268:607-18 (1997); S. Spada et al., Biol.Chem., 378:445-56 (1997); B. Wlotzka et al., Proc. Natl. Acad. Sci.,99:8898-8902 (2002); R. Roberts and J. Szostak, Proc. Natl. Acad. Sci.,94:12297-12302 (1997); P. Colas et al., Proc. Natl. Acad. Sci.,97:13720-25 (2000); and Y. Jiang et al., Anal. Chem., 75:2112-16 (2003).

The term “primers” as used herein refers to oligonucleotides that aredesigned to hybridize with at least one analyte, at least one analytesurrogate, or both, in a sequence-specific manner. Primers typicallyserve as initiation sites for certain amplification techniques,including but not limited to, primer extension and the polymerase chainreaction (PCR).

Probes, according to the teachings herein, are molecules or assembliesthat are designed to combine with at least one analyte, at least oneanalyte surrogate, or both; and can, under appropriate conditions, format least part of at least one molecular complex. Probes typically arepart of at least one probe set, comprising at least one first probe andat least one second probe. In certain embodiments, however, at least oneprobe set can comprise only first probes or second probes, but not bothfirst probes and second probes. In certain embodiments, at least oneprobe of at least one probe set comprises at least one amino acid, atleast one ribonucleotide, at least one deoxyribonucleotide, at least onepeptide nucleic acid (PNA), at least one pseudocomplementary peptidenucleic acid (pcPNA), or combinations thereof.

Probes comprise at least one reaction portion that allow them to bind toor interact with at least one analyte, at least one part of at least oneanalyte, at least one analyte surrogate, at least part of an analytesurrogate, or combinations thereof; typically in a sequence-specific, aconfirmation-specific manner, or both; for example but not limited tonucleic acid hybridization, antigen-antibody binding, aptamer-targetbinding, and the like. In certain embodiments, at least one probe of atleast one probe set further comprises an identity portion or at leastpart of an identity portion comprising at least one coded molecular tag;or an analytical portion or at least part of an analytical portion; buttypically not both an identity portion and an analytical portion. Incertain embodiments, the identity portion is within the reactionportion, coextensive with the reaction portion, or overlaps at leastpart of the reaction portion. In certain embodiments, the analyticalportion is within the reaction portion, coextensive with the reactionportion, or overlaps at least part of the reaction portion.

The reaction portions of nucleic acid probes are of sufficient length topermit specific annealing to complementary sequences in correspondinganalytes, corresponding analyte surrogates, or both; as are primers. Thecriteria for designing sequence-specific nucleic acid probes and primersare well known to persons of ordinary skill in the art. Detaileddescriptions of nucleic acid probe and primer design can be found in,among other places, Diffenbach and Dveksler, PCR Primer, A LaboratoryManual, Cold Spring Harbor Press (1995); Rapley; Schena; and Kwok etal., Nucl. Acid Res. 18:999-1005 (1990). Primer and probe designsoftware programs are also commercially available, for example, PrimerPremier 5, PREMIER Biosoft, Palo Alto, Calif.; Primer Designer 4, Sci-EdSoftware, Durham, N.C.; Primer Detective, ClonTech, Palo Alto, Calif.;Lasergene, DNASTAR, Inc., Madison, Wis.; and iOligo, Caesar Software,Portsmouth, N.H.

In certain embodiments, at least one identity portion, at least part ofthe identity portion, or both comprise at least one coded molecular tagand at least one capture ligand. In certain embodiments, at least oneanalytical portions, at least part of an analytical portion, or both,comprises at least one affinity tag, including but not limited to, atleast one biotin moiety, at least one epitope tag; at least one antibodymolecule; at least one fluorophore; at least one mobility modifier; atleast one hybridization tag; at least one aptamer sequence; orcombinations thereof.

In certain embodiments, at least one probe comprises a reaction portionor part of a reaction portion that is designed to hybridize in asequence-specific manner with a complementary region, i.e., the targetsequences of at least one analyte, at least one analyte surrogate, orboth. In certain embodiments, at least part of the reaction portion ofat least one first probe, at least part of the reaction portion of atleast one corresponding second probe, or both at least part of thereaction portion of the at least one first probe and at least part ofthe reaction portion of the at least one corresponding second probecomprise at least one amino acid, at least one ribonucleotide, at leastone deoxyribonucleotide, at least one PNA, at least one pcPNA, orcombinations thereof.

Typically, the presence of an analyte in a sample can be determinedbased on individually detecting at least one corresponding molecularcomplex or at least part of a corresponding molecular complex, andidentifying the order of the reporter group species. In certainembodiments, the identity of a molecular complex can not be determinedsimply by identifying the order of the reporter group species in thecorresponding identity portion(s). In certain embodiments, the sameidentity portion is used to generate probes for different molecularcomplexes, therefore the identity of such molecular complexes isdetermined by a combination of the order of reporter groups in the codedmolecular tag(s) and at least one additional information element notpresent in the identity portion. For example but not limited to, atleast one reporter group species in the analytical portion (see, e.g.,FIGS. 3 and 4B), at least one reporter group species present in at leastone reaction portion or the combined reaction portions (see, e.g., FIG.4A), or an inherent property of a molecular complex comprising a singleprobe (see, e.g., FIG. 1B). Exemplary inherent properties of suchmolecular complexes include without limitation molecular weight andelectrophoretic mobility. For example but not limited to, a particularcoded molecular tag can be used to assemble one probe species specificfor a small peptide analyte and also to assemble a different probespecies specific for a large protein analyte (the relative molecularweights of the two probes is similar), so that the molecular weight ofthe peptide analyte-molecular complex is substantially less than themolecular weight of the large protein analyte-molecular complex. Thesetwo illustrative molecular complexes can be separated by, for example,size exclusion chromatography. Thus, in this example, the identity ofboth molecular complexes is determined by a combination of the order ofthe reporter group species in the coded molecular tag and the molecularweight of their respective molecular complexes.

The codespace of an identity portion is at least one determinant of thenumber of unique identifier tags or addresses that can be created andcan limit the number of different species of analyte that can bedetermined in a reaction, particularly multiplex reactions. Thetheoretical number of unique identifier tags that can be created withina codespace depends in part on the number of reporter group species tobe used, the properties of those reporter group species, the number ofusable labeling positions in the template, and the detection method(s)employed.

Typically, the template must be large enough so that the reporter groupsat different labeling positions can be individually resolved. In certainembodiments, for individual fluorophores to be optically resolved thelabeling positions are separated by about 0.8 micrometers (μm). Thisspacing exceeds what is typically required to avoid quenching betweenfluorescent reporter groups. Thus, in an exemplary codespace comprising6 labeling positions, a template with a minimum length of about 5 μm istypically needed. In certain embodiments, individual fluorophores to beoptically resolved are separated by about 0.9 μm, about 0.8 μm, about0.7 μm, about 0.6 μm, about 0.5 μm, about 0.4 μm, about 0.3 μm, about0.2 μm, about 0.1 μm, or combinations thereof. The skilled artisan willunderstand that optical resolution depends on several factors includingwithout limitation, the choice of the detection system components andthe distance between the reporter groups because of, among other things,energy transfer between closely positioned fluorescent reporter groups,including quenching and self-quenching.

The skilled artisan will appreciate that the number of unique addressesavailable for identifying molecules of interest, including withoutlimitation, molecular complexes and analytes, can be increased beyondthe number available based on codespace alone. For example, the samecoded molecular tag can be used to form different molecular complexes if(i) they have different affinity portions; (ii) they have differentcapture ligands; (iii) they have labeled reaction portions, includingcolor complementation; or combinations thereof. Additionally, the samecoded molecular tag can be used with the different molecular complexesbased on other differences, including without limitation, the presenceof absence of cleavable linkers; different capture ligands in theidentity portion, the reaction portion, or both; and so forth.

FIG. 4 schematically depicts a multiplicity of distinguishableillustrative molecular complexes, each comprising the same codedmolecular tag RBRB. In FIG. 4A, three biotinylated molecular complexesare shown, each comprising the same coded molecular tag comprising red(R) and blue (B) fluorescent reporter group species in the orderedpattern RBRB and a biotin capture ligand (b). The ligation site is shownas “Λ”, and the combined reaction portions of each molecular complex areshown as “tick” marks between the coded molecular tag and the biotincapture ligand. The combined reaction portions of the upper molecularcomplex lack reporter groups, thus the order of reporter group speciesin the molecular complex is RBRB. The combined reaction portions of themiddle molecular complex comprise the “R” reporter group species, thusthe order of reporter group species in the molecular complex is RBRBR.The combined reaction portions of the bottom molecular complex in FIG.4A comprises both the “R” reporter group species and a green fluorescentreporter group species (G) that by color complementation appear asyellow (“Y”) when individually detected using certain optical SMDtechniques. Thus, the order of reporter group species the bottommolecular complex is RBRBY.

FIG. 4B depicts two molecular complexes, each comprising the same codedmolecular tag comprising the ordered pattern RBRB. The top exemplarymolecular complex in FIG. 4B comprises at least one biotin captureligand (“b”) and the bottom molecular complex comprises at least one DNPcapture ligand (“DNP”). When these two molecular complexes are combinedwith the illustrative patterned substrate, wherein the pattern comprisesone line of anti-biotin antibody capture moieties (shown as α-b) and oneline of anti-DNP antibody capture moieties (shown as α-DNP), the twomolecular complexes are spatially separated on the substrate when theybind their respective capture moieties.

Two different molecular complexes are shown in FIG. 4C, each comprisingthe same coded molecular tag, shown as RBRB, but with differentanalytical portions, here different mobility modifiers. When these twoexemplary molecular complexes are separated using, e.g.,mobility-dependent analytical techniques, they can be isolatedindependently and individually detected. FIG. 4D schematically depictstwo different molecular complexes, each comprising an at least onebiotin moiety and an identity portion comprising the coded molecular tagRBRB and at least one DNP moiety. The top exemplary molecular complexcomprises a cleavable linker between the reaction portion and the codedmolecular tag, while the bottom exemplary molecular complex does notcomprise a cleavable linker. In this illustrative embodiment, the twodifferent molecular complexes are separated using a CaptAvidinchromatography column. The column comprising the bound molecularcomplexes is first treated with an appropriate cleavage reagent torelease the cleavable component comprising the coded molecular tag fromthe top, but not the bottom, molecular complexes. The cleavablecomponents are combined with a first substrate comprising anti-DNPantibody capture moieties and individually detected using an appropriateSMD. The CaptAvidin column is next treated with biotin to reverse theCaptAvidin-biotinylated molecular complex binding, releasing the bottomexemplary molecular complexes. These released molecular complexes arecombined with a second substrate comprising anti-DNP antibody capturemoieties and individually detected using an appropriate SMD.

III. Techniques

Ligation

Ligation according to the present invention comprises any enzymatic orchemical process wherein an inter-nucleotide linkage is formed betweenthe opposing ends of nucleic acid sequences that are adjacentlyhybridized to a template. Additionally, the opposing ends of theannealed nucleic acid probes must be suitable for ligation (suitabilityfor ligation is a function of the ligation method employed). Theinternucleotide linkage can include, but is not limited to,phosphodiester bond formation. Such bond formation can include, withoutlimitation, those created enzymatically by at least one DNA ligase or atleast one RNA ligase, for example but not limited to, T4 DNA ligase, T4RNA ligase, Thermus thermophilus (Tth) ligase, Thermus aquaticus (Taq)DNA ligase, or Pyrococcus furiosus (Pfu) ligase.

Other internucleotide linkages include, without limitation, covalentbond formation between appropriate reactive groups such as between anα-haloacyl group and a phosphothioate group to form athiophosphorylacetylamino group, a phosphorothioate a tosylate or iodidegroup to form a 5′-phosphorothioester, and pyrophosphate linkages.

Chemical ligation can, under appropriate conditions, occur spontaneouslysuch as by autoligation. Alternatively, “activating” or reducing agentscan be used. Examples of activating and reducing agents include, withoutlimitation, carbodiimide, cyanogen bromide (BrCN), imidazole,1-methylimidazole/carbodiimide/cystamine, N-cyanoimidazole,dithiothreitol (DTT) and ultraviolet light.

Ligation generally comprises at least one cycle of ligation, i.e., thesequential procedures of: hybridizing the reaction portions of a firstprobe and a corresponding second probe, that are suitable for ligation,to their respective complementary target regions; ligating the 3′ end ofthe upstream probe with the 5′ end of the downstream probe to form aligation product; and denaturing the nucleic acid duplex to separate theligation product from the analyte or analyte surrogate (see, e.g., FIG.6A). The ligation cycle may or may not be repeated, for example, withoutlimitation, by thermocycling the ligation reaction to linearly amplifythe ligation product that can serve as at least one analyte surrogate.

Also within the scope of the invention are ligation techniques such asgap-filling ligation, including, without limitation, gap-filling OLA andLCR, bridging oligonucleotide ligation, and correction ligation.Descriptions of these techniques can be found, among other places, inU.S. Pat. No. 5,185,243, published European Patent Applications EP320308 and EP 439182, and PCT Publication Nos. WO 90/01069 and WO01/57268.

A “ligation agent”, according to the present invention, can comprise anynumber of enzymatic or chemical (i.e., non-enzymatic) reagents. Forexample, ligase is an enzymatic ligation reagent that, under appropriateconditions, forms phosphodiester bonds between the 3′-OH and the5′-phosphate of adjacent nucleotides in DNA molecules, RNA molecules, orhybrids. Temperature sensitive ligases, include, but are not limited to,bacteriophage T4 ligase and E. coli ligase. Thermostable ligasesinclude, but are not limited to, Taq ligase, Tfl ligase, Tth ligase, TthHB8 ligase, Thermus species AK16D ligase and Pfu ligase. The skilledartisan will appreciate that any number of thermostable ligases,including DNA ligases and RNA ligases, can be obtained from thermophilicor hyperthermophilic organisms, for example, certain species of bacteriaand archaebacteria; and that such ligases can be useful in the methodsand kits of the invention.

Chemical ligation agents include, without limitation, activating,condensing, and reducing agents, such as carbodiimide, cyanogen bromide(BrCN), N-cyanoimidazole, imidazole,1-methylimidazole/carbodiimide/cystamine, dithiothreitol (DTT) andultraviolet light. Autoligation, i.e., spontaneous ligation in theabsence of a ligating agent, is also within the scope of the invention.Detailed protocols for chemical ligation methods and descriptions ofappropriate reactive groups can be found in, among other places, Xu etal., Nucleic Acid Res., 27:875-81 (1999); Gryaznov and Letsinger,Nucleic Acid Res. 21:1403-08 (1993); Gryaznov et al., Nucleic Acid Res.22:2366-69 (1994); Kanaya and Yanagawa, Biochemistry 25:7423-30 (1986);Luebke and Dervan, Nucleic Acids Res. 20:3005-09 (1992); Sievers and vonKiedrowski, Nature 369:221-24 (1994); Liu and Taylor, Nucleic Acids Res.26:3300-04 (1999); Wang and Kool, Nucleic Acids Res. 22:2326-33 (1994);Purmal et al., Nucleic Acids Res. 20:3713-19 (1992); Ashley and Kushlan,Biochemistry 30:2927-33 (1991); Chu and Orgel, Nucleic Acids Res.16:3671-91 (1988); Sokolova et al., FEBS Letters 232:153-55 (1988);Naylor and Gilham, Biochemistry 5:2722-28 (1966); and U.S. Pat. No.5,476,930.

When used in the context of the present invention, “suitable forligation” refers to at least one first probe and at least onecorresponding second probe, wherein each probe comprises anappropriately reactive group based on the ligation reaction employed.Exemplary reactive groups include, but are not limited to, a freehydroxyl group on the 3′ end of the upstream probe and a free phosphategroup on the 5′ end of the downstream probe, phosphorothioate andtosylate or iodide, esters and hydrazide, RC(O)S⁻, haloalkyl, RCH₂S andα-haloacyl, thiophosphoryl and bromoacetoamido groups, andS-pivaloyloxymethyl-4-thiothymidine.

B. Amplification

Amplification according to the present invention encompasses anytechnique by which at least a part of at least one analyte or at leastone analyte surrogate is copied, typically in a template-dependentmanner, including without limitation, a broad range of techniques foramplifying nucleic acid sequences, either linearly or exponentially. Theamplification product of an analyte or part of an analyte is typicallyan analyte surrogate. Exemplary amplification methods include ligasechain reaction (LCR), ligase detection reaction (LDR), polymerase chainreaction (PCR), primer extension, strand displacement amplification(SDA), multiple displacement amplification (MDA), nucleic acidstrand-based amplification (NASBA), and the like, including multiplexversions and combination thereof, for example but not limited to,OLA/PCR, PCR/LDR, PCR/LCR (also known as combined chain reaction-CCR),and the like. Descriptions of such techniques can be found in, amongother places, Sambrook and Russell; Sambrook et al.; Ausbel et al.; PCRPrimer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press(1995); The Electronic Protocol Book, Chang Bioscience (2002)(“TheElectronic Protocol Book”); Msuih et al., J. Clin. Micro. 34:501-07(1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., HumanaPress, Totowa, N.J. (2002)(“Rapley”); U.S. Pat. No. 6,027,998; Barany etal., PCT Publication No. WO 97/31256; Wenz et al., PCT Publication No.WO 01/92579; Ehrlich et al., Science 252:1643-50 (1991); Innis et al.,PCR Protocols: A Guide to Methods and Applications, Academic Press(1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenauet al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin,Development of a Multiplex Ligation Detection Reaction DNA Typing Assay,Sixth International Symposium on Human Identification, 1995 (availableon the world wide web at: promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit Instruction Manual, Cat.#200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci.USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951(1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al.,Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992);Polstra et al., BMC Inf. Dis. 2:18-(2002); and Landegren et al., Science241:1077-80 (1988).

In certain embodiments, amplification comprises at least one cycle ofthe sequential procedures of: hybridizing at least one probe or at leastone primer to target sequences in at least one analyte or at least oneanalyte surrogate; synthesizing at least one strand of nucleotides in atemplate-dependent manner using a polymerase; and denaturing thenewly-formed nucleic acid duplex to separate the strands. The cycle mayor may not be repeated. Amplification methods can comprise thermocyclingor can be performed isothermally. In certain embodiments, at least partof at least one analyte, at least part of an analyte surrogate, orcombinations thereof, is amplified before, during, or after molecularcomplex formation.

In certain embodiments, the methods and kits disclosed herein compriseat least one polymerase, at least one ligation agent, or at least onepolymerase and at least one ligation agent. In certain embodiments,methods comprise ligation reactions; primer extension, including but notlimited to “gap filling” reactions; transcription, including but notlimited to reverse transcription; translation; or combinations thereof,including but not limited to, coupled in vitro transcription/translationsystems.

Primer extension according to the present invention is a process thatcomprises elongating a primer that is annealed to a template in the 5′to 3′ direction using a template-dependent polymerase. According tocertain embodiments, with appropriate buffers, salts, pH, temperature,and nucleotide triphosphates, including analogs thereof, i.e., underappropriate conditions, a polymerase incorporates nucleotidescomplementary to the template strand starting at the 3′-end of anannealed probe or primer, to generate a complementary strand. In certainembodiments, primer extension can be used to fill a gap between twoprobes of a probe set that are hybridized to target sequences of atleast one analyte, at least one analyte surrogate, or combinationsthereof. In certain embodiments, the polymerase used for primerextension lacks or substantially lacks 5′ exonuclease activity.

FIG. 6 schematically depicts exemplary methods for determining thepresence of nucleic acid analytes in a sample, comprising amplification.In FIG. 6A, mRNA analytes (shown as W, M, or L, each with a “poly A”tail) are combined with specific primers (shown as Pr1 or Pr2) andanalyte surrogates comprising single-stranded DNA molecules (shown as M,L, or W, but without poly A tails) are generated by primer extension.Three exemplary probe sets, each comprising one probe with a reactionportion and identity portion comprising a coded molecular tag (shown as123, 321, or 213) and a second probe comprising a reaction portion andan analytical portion comprising DNP are hybridized to the ssDNA analytesurrogates, forming molecular complexes. Ligation product molecularcomplexes are formed in the presence of an appropriate ligation agent.The ligation product molecular complexes are combined with a substratecomprising a patterned surface including anti-DNP antibody capturemoieties and individually detected using an appropriate SMD technique.

C. Separation

Separating comprises any process that removes at least some unreactedcomponents, at least some reagents, or both some unreacted componentsand some reagents from at least one molecular complex, at least part ofat least one molecular complex, or combinations thereof. In certainembodiments, at least one molecular complex, at least part of amolecular complex, or combinations thereof, are separated from unreactedcomponents and reagents, including but not limited to unreactedmolecular species present in the sample, ligation reagents,amplification reagents, for example, but not limited to,unbound/unhybridized probes, primers, enzymes, co-factors, unboundsample components, nucleotides, and the like. The skilled artisan willappreciate that a number of well known separation techniques will beuseful with certain methods disclosed herein.

Exemplary separation techniques include gel electrophoresis, includingbut not limited to isoelectric focusing and capillary electrophoresis;dielectrophoresis; sorting, including but not limited tofluorescence-activated sorting techniques; chromatography, including butnot limited to HPLC, FPLC, size exclusion (gel filtration)chromatography, affinity chromatography, ion exchange chromatography,hydrophobic interaction chromatography, immunoaffinity chromatography,and reverse phase chromatography; ligand-receptor binding, such asbiotin-avidin, biotin-streptavidin, maltose-maltose binding protein(MBP), calcium-calcium binding peptide; aptamer-target binding; zip codehybridization; and the like. Detailed discussion of separationtechniques can be found in, among other places, Rapley; Sambrook et al.;Sambrook and Russell; Ausbel et al.; Molecular Probes Handbook; PierceApplications Handbook; Capillary Electrophoresis: Theory and Practice,P. Grossman and J. Colburn, eds., Academic Press (1992); Wenz andSchroth, PCT International Publication No. WO 01/92579; M. Ladisch,Bioseparations Engineering: Principles, Practice, and Economics, JohnWiley & Sons (2001); and Liebler, Introduction to Proteomics, HumanaPress (2002).

In certain embodiments, separation comprises binding at least onemolecular complex or at least part of a molecular complex to at leastone substrate, either directly or indirectly; for example but notlimited to, indirectly binding a molecular complex or at least part of amolecular complex to a glass substrate, wherein the molecular complexcomprises at least one capture ligand such as biotin, and the substratecomprises at least one capture moiety, such as a streptavidin, avidin,CaptAvidin, or NeutrAvidin; or vice versa. The skilled artisan willunderstand that certain methods comprise at least two differentseparations, for example a first bulk separation that is typically, butneed not be, analytical portion dependent; and a second separationwherein at least one molecular complex comprising at least one captureligand or at least part of a molecular complex comprising at least onecapture ligand is tethered, or attached to a substrate comprising atleast one capture moiety. For example, but without limitation,separating at least one molecular complex or at least part of amolecular complex comprising biotin and at least one mobility modifierby capillary electrophoresis and then tethering or attaching thebiotinylated molecular complex indirectly to a substrate comprisingstreptavidin; or separating at least one molecular complex or at leastpart of a molecular complex comprising an hybridization tag captureligand by RP-HPLC and then indirectly binding the at least one molecularcomplex or at least part of a molecular complex to a glass, mica, orsilicon substrate comprising hybridization tag complement capturemoieties. In certain embodiments, at least one analytical portioncomprises at least one capture ligand, at least one reaction portioncomprises at least one capture ligand, at least one identity portioncomprises at least one capture ligand, or combinations thereof.

In certain embodiments, at least one substrate further comprises atleast one capture moiety. In certain embodiments, at least one substrateis derivatized or coated to enhance the binding of at least one capturemoiety, at least one molecular complex, at least one part of a molecularcomplex, or combinations thereof. Exemplary substrate treatments andcoatings include poly-lysine coating; aldehyde treatment; aminetreatment; epoxide treatment; sulphur-based treatment (e.g.,isothiocyanate, mercapto, thiol); coating with avidin, streptavidin,biotin, or derivatives thereof; and the like. Detailed descriptions ofderivatization techniques and procedures to enhance capture moietybinding can be found in, among other places, Microarray Analysis; G.MacBeath and S. Schreiber, Science 289:1760-63 (2000); A, Talapatra, R.Rouse, and G. Hardiman, Proteogenomics 3:1-10 (2002); Microarray Methodsand Applications-Nuts and Bolts, G. Hardiman, ed., DNA Press (2003); B.Houseman and M. Mrksich, Trends in Biochemistry 20:279-81 (2002); S.Carmichael et al., A Simple Test Method for Covalent Binding MicroarraySurfaces, NoAb BioDiscoveries Microarray Technical Note #010516SC; P.Galvin, An introduction to analysis of differential gene expressionusing DNA microarrays, The European Working Group on CTFR Expression(4-O₂-2003); and Zhu et al., Curr. Opin. Chem. Biol. 7:55-63 (2003). Theskilled artisan will appreciate that lessons learned and techniquesemployed in the nucleic acid and protein microarray arts are generallyapplicable to binding, attaching, or tethering molecular complexes orparts of molecular complexes to substrates. Pretreated substrates andderivatization reagents and kits are commercially available from severalsources, including CEL Associates, Pearland Tex.; Genetix, Ltd.;Molecular Probes, Eugene Oreg.; Quantifoil MicroTools GmbH, JenaGermany; Xenopore Corp., Hawthorne N.J.; NoAb BioDiscoveries,Mississauga, Ontario, Canada; TeleChem International, Sunnyvale, Calif.;CLONTECH Laboratories, Inc., Palo Alto Calif.; Asper Biotech, TartuEstonia; and Accelr8 Technology Corp., Denver Colo. Alternate substratesfor use with the compositions, methods, and kits disclosed herein areProteinPrint™ Films, commercially available from Aspira Biosystems,Inc., So. San Francisco, Calif. In certain embodiments, the substratebound capture moiety comprises at least one amino acid, for example butnot limited to, antibodies, peptide aptamers, peptides, avidin,streptavidin, biotin, and the like. In certain embodiments, thesubstrate bound capture moiety comprises at least one nucleotide, forexample but not limited to, hybridization tag complements, nucleic acidaptamers, PNAs, pcPNAs, and the like.

D. Detection

Detection typically comprises individually detecting at least onemolecular complex or at least part of at least one molecular complex todetermine the presence of the corresponding analyte. Typicallyindividually detecting comprises identifying the order of the reportergroup species in at least one molecular complex or at least part of amolecular complex using at least one SMD technique. The order ofreporter group species is determined collectively, i.e., from an intactor substantially intact coded molecular tag, rather than a group ofdetached subunits or fragments. In certain embodiments, at least onemolecular complex or at least part of a molecular complex isindividually detected while tethered or attached to a substrate via atleast one capture ligand-capture moiety interaction, at least oneelectrostatic interaction, or both. In certain embodiments, at least onemolecular complex or at least part of a molecular complex isindividually detected in solution. In certain embodiments, at least onemolecular complex or at least part of a molecular complex isindividually detected after being isolated on a substrate or in a dilutesolution so that at least one molecular complex or at least part of amolecular complex are spatially separated from other molecular complexesor parts of molecular complexes. The skilled artisan will appreciatethat as the concentration of molecular complexes to be detected in agiven volume or area decreases, the number of spatially separatedmolecular complexes that can be individually detected typicallyincreases.

In certain embodiments, individually detecting comprises optical SMDtechniques that comprise frequency-modulated absorption, laser-inducedfluorescence, or both. The skilled artisan will appreciate that, due tohigh signal-to-noise ratios and low background, laser-inducedfluorescence is frequently used. To reduce background interference, suchas from Raman scattering, Rayleigh scattering, and impurityfluorescence, high-performance optical filters and ultrapure reagentsare typically employed with confocal, near-field, and evanescent wavemicroscopy configurations.

FIG. 5 schematically depicts an exemplary method for individuallydetecting at least one bound molecular complex. In FIG. 5A, an exemplarysubstrate is coated with streptavidin (SA). Two exemplary molecularcomplexes containing an analytical portion comprising at least onebiotin moiety (b) are indirectly tethered to the substrate viabiotin-avidin interactions. The molecular complexes in this examplecomprise identity portions comprising fluorescent reporter groups at sixlabeling positions, GGRBBR (relative to the biotin capture ligand). Thisexemplary molecular complex comprises a reaction portion (not shown)located between the biotin containing analytical portion and theidentity portion. The identity portion comprises a coded molecular tagcomprising a double-stranded DNA template with six reporter groupsattached to labeling positions on the template using PNA and/or pcPNAopeners (including at least one labeling position comprising a PNAopener or a pcPNA opener comprising the “R” reporter group, depicted asX in the enlarged schematic). As shown in FIG. 5B, when the boundmolecular complex is subjected to an elongating force, such as a fluidflow or a field, the molecular complex is stretched in the direction offlow or field. Thus, in this exemplary molecular complex, the analyticalportion serves both as a means for tethering the molecular complex tothe substrate and also orients the identity portion, allowing the orderof the reporter groups in the identity portion to be determined based onthe biotin reference point.

FIG. 5C depicts another exemplary embodiment of the analyte detectionmethods. Here, a cover slip is coated with poly-L-lysine (L), impartinga positive charge (+) to the surface of the cover slip. The molecularcomplex is tethered to the substrate, as before. The molecular complexis stretched due to an elongating or stretching force, shown as a fluidflow or field, the positively charged cover slip surface tends tointeract with the elongated molecular complex at multiple attachmentpoints along its length, attaching it to the cover slip and making iteasier to determine the order of the reporter group species.

FIG. 5D depicts a patterned substrate comprising capture moieties in aseries of parallel lines (for illustration purposes, top to bottom) witha spacing of approximately 20 μm (appropriate for elongating labeled λDNA templates without overlap from one line tethered or attachedmolecular complex to the next). The skilled artisan understands that thedistance between parallel lines on such a patterned substrate can varydepending on the molecular complex or at least part of molecular complexbeing individually detected. Each line of bound capture moieties isshown interacting with molecular complexes, or at least part ofmolecular complexes, comprising identity portions including codedmolecular tags (see the blow up section depicting two identity portionscomprising ordered reporter groups GYYGRY). The left to right arrow atthe bottom indicates a fluid flow or field causing the indirectly boundmolecular complexes or at least part of molecular complexes to elongatedue to the flow or field. The tethered or attached molecular complexesor at least part of molecular complexes are individually detected usingan appropriate SMD to determine the order of the reporter groups in eachidentity portion.

In certain embodiments, individually detecting comprises opticaldetection of at least one molecular complex in solution. In certainembodiments, solution phase optical detection comprises timed-gatedfluorescence. In certain embodiments, optical detection comprises atleast one electrophoresis capillary, including without limitation,microcapillaries and nanocapillaries; at least one sheath flow; at leastone microfluidic device; or combinations thereof, wherein molecularcomplexes or at least parts of molecular complexes are individuallydetected and the order of the corresponding reporter group species isidentified. In certain embodiments, individually detecting comprisesdetecting at least one molecular complex or at least part of a molecularcomplex in at least one microdroplet. In certain embodiments, at leastone electrodynamic trap is used to levitate at least one microdropcomprising at least one molecular complex or at least part of amolecular complex. Detailed descriptions of SMD techniques forindividually detecting at least one molecular complex or at least partof a molecular complex in solution can be found in, among other places,Single Molecule Detection in Solution: Methods and Applications, C.Zander, J. Enderlein, and R. Keller, eds., John Wiley & Sons, Inc.(2002); M. Barnes et al., Anal. Chem. 67:A418-23 (1995); M. Barnes etal., J. Opt. Soc. Am. B 11:1297-1304 (1994); S. Nie and R. Zare, Ann.Rev. Biophys. Biomol. Struct. 26:567-96 (1997); M. Foquet et al., Anal.Chem. 74:1415-22 (2002); S. Weiss, Science 283:1676-83 (1999); C.-Y.Kung et al., Anal. Chem. 70:658-661 (1998); M. Wabuyele et al.,Electrophoresis 22:3939-3948 (2001); W. Ambrose et al., Chem. Rev.99:2929-56 (1999); P. Goodwin et al., Acc. Chem. Res. 29:607-13 (1996);and R. Keller et al., Anal. Chem. 74:316A-24A (2002).

The detection and decoding techniques used for solution phasefluorescence detection formats typically have similar fluorescence andspatial resolution concerns as bound, i.e., tethered or attached,fluorescence detection formats. In both formats, fluorescence, whetherfrom an individual molecular complex or a cleavable component, istypically detected on detectors with appropriate optical filters or animaging spectrograph. Orientation in bound detection formats istypically based on the tether or attachment points, but can be based onparticular coding patterns, for example but not limited, a particularreporter group is always used in a specified labeling position andnowhere else.

Alignment of solution phase molecular complexes or parts of molecularcomplexes can be achieved by, for example but not limited to, flow alonga capillary, between plates with a narrow gap, or through an appropriatemicrofluidic device. The flow stream can align the molecular complexesor parts of molecular complexes by, for example but not limited to,sheath flow, microfluidic channel structures, or by solvent polymerinteractions. In certain embodiments, the flow velocity is designed toinsure that a molecular complex or a part of a molecular complex spendsa specified amount of time in the detection region, that only onemolecular complex or cleavage component is present in the detectionregion at a given time, or both.

Orientation of solution phase molecular complexes or parts of molecularcomplexes for identifying the order of their corresponding reportergroups can be achieved using particular coding patterns, for example butnot limited, a particular reporter group species is always used in aspecified labeling position and nowhere else or a fixed, identifiablereporter group order at two or more specific labeling positions andvarying reporter group species at one or more of the other labelingpositions. Flow cells with single molecule channels can be used andindividual molecular complexes or cleavable components can be forcedinto such channels for orientation during detection and decoding, usingfor example but not limited to, a multi-spectral analog detector or animaging detector. In certain embodiments, multiple solution phasemolecular complexes, parts of molecular complexes, or both, are passedthrough a wide channel and multi-spectral images are taken for analyzingand decoding the order of reporter group species of individuallydetected molecular complexes and/or parts of molecular complexes.

In certain embodiments, individually detecting comprises near fieldmicroscopy, including but not limited to near-field scanning opticalmicroscopy; far-field microscopy, including but not limited to,far-field confocal microscopy and fluorescence-correlation spectroscopy;wide-field epi-illumination microscopy, evanescent wave excitationmicroscopy or total internal reflectance (TIR) microscopy; scanningconfocal fluorescence microscopy; the multiparameter fluorescencedetection (MFD) technique; two-photon excitation microscopy; orcombinations thereof. In certain embodiments, individually detectingcomprises fluorescence detection integrated with atomic-forcemicroscopy, for example but not limited to, using an inverted opticalmicroscope; or fluorescence excitation spectroscopy combined withshear-force microscopy. Detailed descriptions of such techniques can befound in, among other places, S. Nie and R. Zare, Ann. Rev. Biophys.Biomol. Struct. 26:567-96 (1997); R. Brown et al., Review of SingleMolecule Detection in Biological Applications, NPL Report COAM 2,National Physics Laboratory, Middlesex, United Kingdom (2001)(“Brown etal.”); P. Rothwell et al., Proc. Natl. Acad. Sci. 100:1655-60 (2003); C.Eggeling et al., J. Biotechnol. 86:163-80 (2001); W. Ambrose et al.,Chem. Rev. 99:2929-56 (1999); S. Weiss, Science 283:1676-83 (1999); G.Segers-Nolten et al., Nucl. Acid Res. 30:4720-27 (2002); and J.Michaelis et al., Nature 405:325-28 (2000).

In certain embodiments, individually detecting comprises scanning probemicroscopy techniques, applied optical spectroscopy techniques,nanoelectromechanical (NEMS) techniques, or combinations thereof. Incertain embodiments, individually detecting comprises at least one ofthe following SMD techniques: scanning tunneling microscopy; atomicforce microscopy (AFM), including but not limited to cryo-AFM andsingle-walled carbon nanotube-AFM (SWNT-AFM); spectrally resolvedfluorescence imaging microscopy (SFLIM); surface enhanced Ramanspectroscopy (SERS); surface enhanced resonant Raman spectroscopy(SERRS); surface plasmon resonance (SPR); and scanning electrochemicalmicroscopy (SECM). Detailed descriptions of such SMD techniques can befound in, among other places, Brown et al., and Woolley et al., Nat.Biotechnol. 18:760-63 (2000).

In certain embodiments, at least one molecular complex or at least apart of a molecular complex interacts with or becomes attached ortethered, directly or indirectly, to a substrate by one or moreattachment points. In certain embodiments, at least one substratecomprises one or more surfaces to which a molecular complex or at leastpart of a molecular complex can interact, become attached or tethered,either directly or indirectly. For example, but not limited to,non-covalent attachment, such as by hybridization with at least onehybridization tag complement, capture moiety-capture ligand interaction,aptamer-target binding, electrostatic interaction, hydrophobicinteraction, nonspecific adsorption, solvent evaporation, on or in apolymer such as a hydrogel, such as agarose, polyacrylamide, or thelike; on or in a spin cast polymer coating, such as apolylmethylmethacrylate (PMMA) coat (e.g., M. Prummer et al., Anal.Chem. 72:443-47 (2000)); and the like. In certain embodiments,substrates are used to enhance individual detection of at least part ofa molecular complex. For example but not limited to, tethering amolecular complex or at least part of a molecular complex in a fluidflow or electric or dielectric field to provide an orientation orreference point for determining the order of reporter group species.

Substrate surfaces are typically planar, but can comprise a wide varietyof topographies, including without limitation, concave, convex, andcombinations of topographies on the same surface. Substrates for opticaldetection are typically composed of materials that are preferably (i)optically transparent, (ii) minimally reflective, (iii) minimallyabsorptive, and (iv) low fluorescence. The skilled artisan willunderstand that if optical detection comprises visualization from thesame side as the illumination, then the substrate may, but need not beoptically transparent. Exemplary substrates can be composed of one ormore of the following: glass, including but not limited to borosilicateglass; quartz, including but not limited to fused quartz; mica;plastics, including but not limited to polystyrene, polycarbonate,polymethacrylate (PMA), PMMA, polydimethylsiloxane (PDMS); silicon,including silica-containing materials; germanium; graphite; films,including but not limited to, gold film, silver film, aluminum film,diamond film; and the like. The skilled artisan will appreciate that thesuitability of a particular substrate, including its topography andcomposition, typically depends at least in part on the detectiontechnique to be employed.

In certain embodiments, the substrate is pretreated, including but notlimited to activation and/or derivatization treatments. Substrates canbe derivatized or activated, for example but not limited to treatmentwith polylysine and various silanes, such as trimethoxysilanes,aminosilanes, including but not limited to APTES, to produce among otherthings, amine surfaces or aldehyde surfaces. These derivatized surfacesallow various capture moieties to be attached or tethered to thesubstrate. In certain embodiments, capture moieties are dried orevaporated onto a substrate. In certain embodiments, oligonucleotidecapture moieties comprising, for example but not limited to3′-propanol-derivitized residues or 5′-disulfide modifications, aredirectly coupled to underivatized substrates. In certain embodiments,such oligonucleotides are functionalized at their 5′ terminus withactivated 1-O-mimethoxytrityl hexyl disulfide1′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (Rogers et al.,Anal. Biochem. 266:23 et seq., (1999)). Such disulfide bridge linkedcapture moieties can be cleaved by reducing agents. In certainembodiments, a molecular complex or at least part of a molecular complexbound to a capture moiety comprising such disulfide bridges is releasedfrom a substrate under reducing conditions. Detailed descriptions ofsubstrates, substrate activation methods, and the like, can be found in,among other places, Beaucage, Curr. Clin. Med. 8:1213-44 (2001); Diehlet al., Nucl. Acid Res. 29, No. 7 e38, pages 1-5 (2001); MicroarrayAnalysis; DNA Microarrays A Practical Approach, M. Schena, ed., OxfordUniversity Press (1999); R. Stears et al., Nature Medicine 9:140-145(2003), including all Supplementary Tables and the Supplementary Note;and DNA Array Image Analysis Nuts & Bolts, G. Kamberova and S. Shah,eds., DNA Press, LLC (2002).

IV. Exemplary Embodiments

A. Coded Molecular Tag Fabrication

According to the teachings herein, coded molecular tags can befabricated using a variety of methods, including without limitation,template-independent subunit assembly, template-dependent subunitassembly, and template-dependent subunit synthesis.

In certain embodiments, coded molecular tags are fabricated using asingle-stranded nucleic acid template of known sequence and a series ofreporter group-labeled oligonucleotides designed to anneal tocomplementary sites on the template. The labeled oligonucleotides areannealed to the template providing an ordered pattern of reportergroups, as shown in FIG. 12A. In certain embodiments, there are gapsbetween the labeled-oligonucleotides that are annealed to the template.In certain embodiments, coded molecular tag fabrication methods furthercomprise gap-filling primer extension, as shown in FIG. 13. In certainembodiments, methods for fabricating coded molecular tag compriseligation. In certain embodiments, series of contiguous or nearlycontiguous synthetic primers labeled with reporter group specieshybridize contiguously to a single-stranded nucleic acid template.Additional primers labeled with a different reporter group species or aseries of primers labeled with a different reporter group species can behybridized either simultaneously or subsequently to the single-strandedtemplate. In certain embodiments, such hybridized labeled primers areligated together under appropriate conditions. In certain embodiments,at least one primer is extended by primer extension, then ligated, asshown in FIG. 13.

In certain embodiments, at least one coded molecular tag is fabricatedusing a stepwise primer extension process. As shown in FIG. 13, at leastone primer pair comprising a start primer (shown as Pr1 in FIG. 13) anda stop primer (shown as Pr2 in FIG. 13) is hybridized with asingle-stranded template to form a hybridization complex. In certainembodiments, the stop primer is non-extendable, i.e., it can not beextended by a polymerase in a primer extension reaction, e.g., itcomprises a dideoxynucleotide on its 3′ end. In the presence of anappropriate polymerase and under appropriate conditions, including atleast one labeled nucleotide triphosphate, the start primer is extendedto the vicinity of the stop primer by primer extension and at least onenucleotide comprising a reporter group (shown as C-1, T-2, and G-3 inFIG. 13) is incorporated in the newly synthesized section. In certainembodiments, the hybridization complex comprising at least one newlysynthesized labeled section is heated to denature the stop primer.Additional primer pairs are hybridized to single-stranded regions of thetemplate (shown as Pr3 and Pr4 in FIG. 13) and the process repeated asnecessary to fabricate a semi-synthetic coded molecular tag comprising amultiplicity of synthesized subunits comprising reporter group species.The illustrative coded molecular tag shown in FIG. 13 further comprisesan oligonucleotide adapter and a single-stranded overhanging 3′ end.Such adaptors and overhanging ends are useful for, among other things,combining coded molecular tags and assembling probes.

In certain embodiments, the primers and synthesized sections of suchcoded molecular tags are ligated together. In certain embodiments, twoor more primer pairs are hybridized to the same single-stranded templateand the same reporter group species is incorporated into multiplelabeling positions in parallel during the same primer extensionreaction. In certain embodiments, coded molecular tags comprise at leastone nucleotide adapter, for example but not limited to, anoligonucleotide linker.

In certain embodiments, coded molecular tags are fabricated using codedmolecular tag subunits comprising at least two restriction fragmentsthat are ligated together. In certain embodiments, the individualrestriction fragments are labeled with reporter species usingintercalating agents, as described in Example 2 (see also FIG. 7A or7B). In certain embodiments, the restriction fragments are labeled usingsynthetic methods, for example without limitation, as described inExample 3. In certain embodiments, the restriction fragment arechemically-labeled, enzymatically-labeled, or both (see, e.g., Examples3 and 4).

Coded molecular tags can be fabricated using coded molecular tagsubunits, including without limitation, reporter group-labeled PCRplasmid DNA with engineered cohesive ends. In one exemplary embodiment,shown in FIG. 7B, two aliquots of this plasmid are PCR amplifiedseparately, using different sets of forward and reverse primers withtails comprising restriction enzyme cleavage sites for PacI, or NotI, orPsiI (shown as arrows labeled “PacI”, “NotI”, or PsiI). The resultinglinear double-stranded PCR amplicons each has either a PacI linker and aNotI linker at its ends, or a PsiI and a NotI linker at its ends. Theamplicon on the left has the PacI linker on its left end and the NotIlinker on its right end, while the amplicon on the right has the NotIlinker on its left end and the PsiI linker on its right end. Theamplicons are separately labeled using intercalating dyes,chemical-labeling, or enzymatic-labeling methods, forming codedmolecular tag subunits. The two coded molecular tag subunits are cleavedusing restriction enzymes Pac I and Not I for the coded subunit on theleft, and using restriction enzymes PstI and Not I for the coded subuniton the right in order to form cohesive ends. Then the two codedmolecular tag subunits are combined, annealed and ligated to form acoded molecular tag comprising two ordered reporter group species. Theskilled artisan will understand that the directional ligation techniqueused here is helpful to limiting self-ligation during the fabrication ofcoded molecular tags.

In certain embodiments, coded molecular tags are fabricated using atleast one synthetic subunit comprising at least one reporter group. Asshown in FIG. 12A, a single stranded piece of nucleic acid of knownsequence is used as a template. A series of contiguous oligonucleotides(oligos) are synthesized based on sequence of the template such thatwhen hybridized with the template, essentially all of the templatebecomes double-stranded except for a short single stranded tail on oneend. The synthetic oligos are labeled with reporter groups as follows:the first set of contiguous oligos comprise at least some incorporatednucleotides labeled with reporter group R; the second set of contiguoussynthetic oligos comprise at least some incorporated nucleotides labeledwith reporter group B. When each of these synthetic oligos arehybridized to the template, a double-stranded coded molecular tag isformed comprising reporter groups in the order RB. This illustrativecoded molecular tag can be ligated together in the presence of anappropriate ligation agent. A gap-filling step may be employed prior toligation if at least some of the labeled oligos are not contiguous.

The skilled artisan will understand that such nucleic acid-based codedmolecular tag fabrication methods will work with essentially any nucleicacid template of appropriate length and with oligonucleotides of varyinglengths, for example but not limited to, about 25 nucleotides long,about 30 nucleotides long, about 40 nucleotides long, about 45nucleotides long, about 50 nucleotides long, about 60 nucleotides long,or even longer if their synthesis is feasible. If longer labelingpositions are desired, additional contiguous oligonucleotides can belabeled with the appropriate reporter group or larger syntheticoligonucleotides can be used, or both. If spaces are desired between thelabeling positions, unlabeled oligonucleotides of the desired length canbe hybridized between the labeling positions.

In certain embodiments, coded molecular tags are fabricated with orderedgroups not comprising fluorophores, including without limitation,non-fluorophore affinity tags, as shown in FIG. 12B. Such probes can besubsequently labeled with fluorophores, if desired, using appropriatefluorophore-labeled anti-affinity tag antibodies, as shown in FIG. 12B.The skilled artisan will understand that the order of fluorescentreporter groups in such coded molecular tags is determined, in part, bythe labeled antibodies being used. For example, to obtain the orderfluorescein-rhodamine-Texas Red-Oregon Green using the coded moleculartag depicted in FIG. 12B, the following antibodies would be used:fluorescein-labeled anti-c-Myc antibody, rhodamine-labeled anti-DNPantibody, Texas Red-labeled anti-Penta-His antibody, and OregonGreen-labeled anti-VSV-G antibody.

In certain embodiments, coded molecular tags are fabricated usingdouble-stranded reporter group-labeled synthetic oligonucleotides thatare ligated together in a desired order. As shown in FIG. 12C, fivecoded molecular tag subunits (depicted as 1, 2, 3, 4, and 5 in FIG. 12C)are synthesized with appropriate cohesive ends. Subunits 1 and 2comprise reporter group B, subunits 3 and 4 comprise reporter group R,and subunit 5 comprises reporter group G. When these subunits arecombined, either collectively or in a step-wise manner, they will annealprovided that they possess appropriate cohesive ends forming a codedmolecular tag. In the presence of an appropriate ligation agent, such asligase, the annealed coded molecular tag subunits are ligated. Theseexemplary coded molecular tag subunits are designed so that theiroverhanging ends can serve cohesive ends for annealing desiredoligonucleotides together. By annealing appropriately labeled syntheticoligonucleotides together, coded molecular tags comprising reportergroup species in ordered patterns can be fabricated. Optionally, theannealed oligonucleotides can be ligated using a ligation agent. Suchoverhanging ends on these exemplary coded molecular tag subunits canalso facilitate, among other things, annealing smaller coded moleculartags to generate larger coded molecular tags and probe assembly. Theskilled artisan appreciates that overhanging ends can be located on the5′ end(s), the 3″ end(s), or both and can be synthesized with anydesired sequence.

In certain embodiments, coded molecular tags are fabricated using asingle-stranded nucleic acid template comprising a sequence designed toallow incorporation of reporter-group labeled nucleotides only atspecific locations. For example without limitation, a synthetic templatecomprising the artificial sequenceGTTGT(T)_(n)TATTAT(T)_(n)TCTTCT(T)_(n)TGCTTAA (SEQ ID NO.: 1) iscombined with a primer comprising the sequence TTAAGC, an appropriatepolymerase, unlabeled dATP, and dCTP, dGTP, and dTTP, labeled withreporter groups 1, 2, and 3 respectively. Under appropriate conditions,a double-stranded nucleic acid coded molecular tag is generated byprimer extension, wherein the nascent strand comprises the sequenceTTMGCA(A)_(n)AG(2)AAG(2)A(A)_(n)AT(3)MT(3)A(A)_(n)AC(1)AAC(1) (SEQ IDNO:2) with the ordered reporter group pattern 2-3-1. For opticaldetection methods, labeling positions are typically about 1 μm or moreapart, or for nucleic acid coded molecular tags, about 3000 bases ormore apart. Thus, in certain embodiments, (T)_(n) and (A)_(n) compriseabout 3000 Ts or 3000 As, respectively; about 3500 Ts or As,respectively; about 4000 Ts or As, respectively; about 5000 Ts or As,respectively; or about 10000 Ts or As respectively.

The skilled artisan will appreciate that coded molecular tags can be canbe mass-produced and stored for use as “off the shelf” interchangeablecomponents for assembling probes for specific applications. In certainembodiments, templates and/or coded molecular tags further comprise oneor more cleavable linker group; one or more restriction enzyme siteand/or adapter sequence to facilitate, among other things, the assemblyof probes; one or more affinity tag, aptamer sequence, or hybridizationtag for separation and/or substrate attachment or tethering procedures;and combinations thereof. In certain embodiments, at least one reportergroup is attached to at least one template with a PNA and/or pcPNAopener, clamp, earring structure, or the like (see, e.g., O. Zelphati etal., BioTechniques 28:304-16 (2000); Demidov et al., Methods 23:123-31(2001); Izvolsky et al., Biochemistry 39:10908-13 (2000); Lohse et al.,Proc. Natl. Acad. Sci. 96:11804-08 (1999); and Kuhn et al., J. Amer.Chem. Soc. 124:1097-1103 (2002)).

B. Probe Assembly.

Probes, according to the disclosed teachings, are molecules orassemblies that are designed to combine with at least one analyte, atleast one analyte surrogate, or both, typically forming at least part ofat least one molecular complex. Probes comprise at least one reactionportion that allows them to bind to or interact with at least oneanalyte, at least one part of at least one analyte, at least one analytesurrogate, at least part of an analyte surrogate, or combinationsthereof, typically in a sequence-specific or confirmation-specificmanner, for example but not limited to, nucleic acid hybridization,antigen-antibody binding, aptamer-target binding, and the like. Theskilled artisan will understand that probes comprising at least onecoded molecular tag can be assembled using a variety of methods known inthe art, for example, without limitation, ligation techniques andcrosslinking techniques (see, e.g., Example 9). Detailed descriptions ofsuch procedures can be found in, among other places, Maniatis et al.;Sambrook et al.; Sambrook and Russell; Ausbel et al.; BioconjugateTechniques; and The Electronic Protocol Book. In certain embodiments, atleast one DNA coded molecular tag comprises at least one phosphorylatedlinker, at least one non-phosphorylated linker, at least one adapter, orcombinations thereof (collectively, “adapters”; see, e.g., New EnglandBioLabs 2002-03 Catalog & Technical Reference, particularly at pages142-145, New England BioLabs, Inc., Beverly, Mass.; Stratagene 2003/2004Catalog, particularly at page 211).

Exemplary probe assembly methods comprising ligation are shownschematically in FIG. 8. In FIG. 8A, a coded molecular tag comprisingthe ordered reporter group species RBBY and an illustrativesingle-stranded linker comprising the nucleotide sequence “cctg” iscombined with an exemplary ligation template comprising the nucleotidesequence “ggaccagg” and a single-stranded oligonucleotide comprising thesequence “gtccxxxxx”. These illustrative probe components are annealedand then ligated to generate a probe comprising an identity portionincluding a coded molecular tag and a reaction portion (shown as “xxxxx”in the probe; the sequence and/or length of the reaction portionsvaries, in part, due to the target sequences on the correspondinganalyte or analyte surrogate). FIG. 8B depicts another exemplary probeassembly method, wherein a DNA coded molecular tag comprising orderedreporter groups BYBØR, where Ø represents a labeling site that isvacant, and a linker with the nucleotide sequence “tatat”, is combinedwith an oligonucleotide comprising the sequence “atataxxxx” (shown as“OLIGO”). These illustrative probe components are annealed and ligatedto generate a probe comprising an identity portion and a reactionportion (shown as the variable sequence “xxxx” in the probe).

In certain embodiments, probes of the invention are assembled usingcoded molecular tags. In certain embodiments, at least one codedmolecular tag is incorporated in at least one identity portion. Incertain embodiments, at least one first probe, at least one secondprobe, or at least one first probe and at least one second probecomprise at least one coded molecular tag. In certain embodiments, atleast one coded molecular tag is coupled, either covalently ornon-covalently, to an adapter such as a nucleotide linker sequence, asshown in FIG. 13, and as described in Example 3. In certain embodiments,the adapter facilitates the incorporation of at least one codedmolecular tag (see, e.g., FIG. 8). In certain embodiments, at least onecoded molecular tag comprises at least one capture ligand (see, e.g.,FIG. 3, panel F). In certain embodiments, at least one coupled codedmolecular tag-adapter comprises at least one capture ligand.

In certain embodiments, at least one adapter is located near or in thecoded molecular tag so that it is: (i) at or near one end of the orderedreporter group species and/or (ii) near at least one capture ligand tofacilitate attachment or tethering of at least one probe. In certainembodiments, cleavage at one or more restriction enzyme cleavage siteswithin an adapter generates blunt ends, releasing at least one cleavablecomponent. In certain embodiments, cleavage at one or more restrictionenzyme cleavage sites within an adapter generates cohesive ends that canfacilitate annealing and ligation during coded molecular tagfabrication, probe assembly, or both, as shown in FIG. 7B.

In certain embodiments, probe assembly comprises ligating at least onecoded molecular tag to at least one oligonucleotide comprising at leastone reaction portion using an appropriate ligation template, such as theillustrative ligation template shown in FIG. 8A, to generate a exemplaryprobe comprising at least one reaction portion and an identity portion.The skilled artisan will appreciate that the ligation template may, butneed not be, part of the probe. In other embodiments, a coded moleculartag is combined with an oligonucleotide comprising “cohesive ends”, forexample as shown in panel FIG. 8B. The two sequences can anneal underappropriate conditions, forming a probe, as shown in FIG. 8B. Theannealed duplex can be ligated together, under appropriate conditions,using at least one ligation agent.

In certain embodiments, at least one probe comprising at least oneidentity portion forms a molecular complex with an analyte or an analytesurrogate in a multiplex reaction format. At least one molecular complexor at least part of a molecular complex is separated using, for examplebut not limited to, electrophoretic, chromatographic and/or affinityseparation techniques. At least one separated molecular complex or atleast part of a molecular complex is individually detected and thepresence of the corresponding analyte is determined. In certainembodiments, at least one probe further comprises at least one cleavablecomponent comprising at least part of an identity portion. In certainembodiments, at least one probe further comprises at least one cleavablecrosslinker. In certain embodiments, cleavage of at least onecrosslinker releases at least one cleavable component from at least onemolecular complex or at least part of a molecular complex. The skilledartisan understands that a cleavable component is included within theterm “part of a molecular complex.”

In certain embodiments, at least one cleavable component comprising atleast part of an identity portion further comprises at least one captureligand (see, e.g., FIG. 4D). In certain embodiments, at least onecleavable component comprising at least one identity portion or at leastpart of an identity portion, further comprises at least one affinitytag, at least one aptamer, at least one hybridization tag, orcombinations thereof. The skilled artisan will appreciate that incertain embodiments, the cleavable components containing at least partof a molecular complex are similar in concept to the cleavableisotope-coded affinity tags (ICAT; Applied Biosystems) used in some massspectroscopy applications (see, e.g., Gygi et al., Nature Biotech.17:994-44 (1999) and that mass spectral reporter groups are also withinthe scope of the invention.

Crosslinkers, typically join two or more molecules, by a covalent bond.Crosslinking reagents usually contain two reactive groups, for examplebut not limited to, succinimidyl esters, maleimides, and iodoacetamides,that may be the same (homobifunctional) or different(heterobifunctional). The reactive groups participate in covalent bondformation during chemical, thermal, or photo-activated reactions.Crosslinkers are referred to as cleavable or non-cleavable, dependent ontheir chemical composition and/or photolability. Cleavable crosslinkerscan be cleaved into at least two parts, depending on their composition,when exposed to appropriate conditions and/or reagents for example butnot limited to, cleavage of disulfides by reducing agents; cleavage ofglycols and diols by periodates; diazo linkages cleaved by dithionate;ester linkages cleaved by hydroxylamine; sulfone linkages cleaved bybases; and the like. Crosslinking reagents, including cleavablecrosslinkers, are available from several commercial sources, includingPierce Biotechnology, Inc., Rockford Ill.; and Molecular Probes, Inc.,Eugene Oreg. Photocleavable compounds or photocleavable elementsincorporated into at least one probe, at least one molecular complex, orboth, are expressly within the intended scope of the invention. Incertain embodiments, under appropriate photocleavage conditions at leastone cleavable component is obtained from at least one molecular complexor at least part of a molecular complex. Detailed descriptions ofcrosslinkers and their use can be found in, among other places, Pierce2003-2004 Applications Handbook & Catalog, Pierce Biotechnology, Inc.(2003)(“Pierce Applications Handbook”); Handbook of Fluorescent Probesand Research Products, 9^(th) ed., Molecular Probes, Inc.(2002)(“Molecular Probes Handbook”); DOUBLE AGENTS™ Cross-LinkingReagents Selection Guide, Pierce Chemical Co. (2001); BioconjugateTechniques; S. Verma and F. Eckstein, Ann. Rev. Biochem. 67:99-134(1997) and the Glen Research 2002 Catalog.

In certain embodiments, at least one probe set comprises at least oneantibody molecule that reacts specifically with at least one analyte, atleast one analyte surrogate, or both. In certain embodiments, at leastone probe set comprises at least one aptamer that reacts specificallywith at least one analyte, at least one analyte surrogate, or both.Certain embodiments of the compositions, methods, and kits furthercomprise at least one antibody molecule, at least one aptamer, or both,that specifically react with at least one first probe, at least onesecond probe, at least one molecular complex, at least part of amolecular complex, at least one capture moiety, at least one captureligand, or combinations thereof.

C. Analyte Detection

1. Molecular Complex Formation.

In certain embodiments, one or more probe can hybridize with or bind toat least one analyte, at least one analyte surrogate, or combinationsthereof, to form a molecular complex. In certain embodiments, at leastone first reaction portion of at least one first probe and at least onesecond reaction portion of at least one corresponding second probe aredesigned to hybridize to complementary “target” sequences on the samestrand of at least one analyte, at least one analyte surrogate, orcombinations thereof. In certain embodiments, the probes in at least oneprobe set are suitable for ligating together when hybridized adjacent toone another (see, e.g., FIG. 1A, 1:1P1:2P1A). In certain embodiments, atleast one first probe and at least one corresponding second probe aredesigned to hybridize to the same strand of at least one analyte or atleast one reaction intermediate, at least one analyte surrogate, orboth, but they do not hybridize adjacent to each other (see, e.g., FIG.1A, 2:1P2:2P2B). In certain embodiments, the probes of at least oneprobe set are designed to hybridize to opposite strands of at least oneanalyte, at least one analyte surrogate, or both.

In certain embodiments, molecular complexes comprise at least oneligation product resulting from the ligation of at least one first probeand at least one corresponding second probe, as shown schematically inFIG. 1A. In certain embodiments, such ligation product molecularcomplexes further comprise at least one analytical portion (see FIGS. 1Aand 3). In certain embodiments, the first probe and the second probefrom the same probe set hybridized adjacent to each other. In certainembodiments, the first probe and the second probe do not hybridizeadjacent to each other, but the 3′ end of the 5′ (upstream) probe isextended, under appropriate conditions and in the presence of at leastone polymerase, until the extended 3′ end of the upstream probe isadjacent to the downstream probe, sometimes referred to as “gap-filling”(see, e.g., FIG. 1A, 2:1P2:2P2B). In the presence of at least oneligation reagent and under appropriate conditions, at least one ligationproduct molecular complex is formed.

In certain embodiments, at least one molecular complex comprises atleast one analyte surrogate, at least part of an analyte surrogate, atleast one analytical portion, or combinations thereof (see, e.g., FIG.6). In certain embodiments, at least one analyte surrogate comprises atleast one nucleotide (see, e.g., FIG. 6A) or at least one amino acid(see, e.g., FIG. 6B).

In certain embodiments, at least one molecular complex comprises atleast one probe and at least one analyte, wherein the at least one probeand the at least one corresponding analyte specifically interact but donot “hybridize” (see, e.g., FIG. 1B). For example but not limited to, aninsulin molecule bound to at least one anti-insulin antibody comprisinga coded molecular tag; a viral antigen such as hepatitis B surfaceantigen (HBsAg) and at least one anti-HBsAg antibody comprising a codedmolecular tag; or the like. In certain embodiments such molecularcomplexes further comprises at least one analytical portion.

2. Nucleic Acid Analytes.

The disclosed compositions, methods, and kits can be used in a widevariety of applications to determine the presence of nucleic acidanalytes in a sample. For example, the compositions, methods, and kitsdisclosed herein are useful for gene sequence analyses such asgenotyping applications, including but not limited to sequenceevaluation for SNP detection and identification; gene expressionapplications, including but not limited to mRNA expression profiling,splice variant analyses, and gene expression modification analyses,including but not limited to gene knock-down, gene knock-out, geneknock-in, gene up-regulation, gene down-regulation, and the like; ncRNAstudies; mutation analyses including without limitation, evaluatingheritable and somatic mutations; evaluating drug-resistant mutants inparasites, microorganisms, and viruses; and the like.

FIG. 1A depicts exemplary probes and methods for determining thepresence of nucleic acid analytes. The upper panel of FIG. 1A depicts asample comprising three molecular species, designated 1, 2, and 3,wherein species 1 and 2 represent analytes of interest. This sample ismixed with exemplary probe sets one and two, designed to determine thepresence of analyte species 1 and 2. The probe set for molecular species1 comprises three types of probes, a first probe (1P1) comprising areaction portion and an identity portion comprising reporter groups Rand G in the ordered sequence RGRG (left to right). The first probe setalso comprises two species of second probe, designated 2P1A and 2P1B,each comprising a reaction portion and an analytical portion, butdiffering in the sequence of their respective reactive portions so thatmost frequently only one second probe fully hybridizes withcomplementary sequences of analyte 1 under appropriate reactionconditions. When properly annealed with analyte 1, the two probe speciesof probe set one hybridize adjacently (shown as 1:1P1:2P1A). The secondprobe set also comprises three probe species, one first probe (1P2),comprising an analytical portion, and two second probe species,designated 2P2A and 2P2B. Both of these second probes comprise anidentity portion comprising reporter groups R and G, but positioned indifferent orders, so the order of 2P2A is RRRR, and the order for 2P2Bis GGRG. When properly annealed with analyte 2, the two probe speciesare hybridized to the same strand of analyte 2 (shown schematically as2:1P2:2P2B), but they are not hybridized adjacently due to a gap betweenthe 5′ end of the annealed second probe (here, 2P2B) and the 3′ end ofthe first probe (shown schematically as 1P2). Under appropriateconditions, e.g., in the presence of at least one appropriatepolymerase, nucleotide triphosphates, salts, and reaction conditions,the gap between the hybridized probes of the second probe set is closedby primer extension. In the presence of an appropriate ligation reagentand under suitable conditions, the annealed probes of both the firstprobe set and the second probe set are ligated together to form ligationproduct molecular complexes 1 and 2, respectively (shown hybridized totheir corresponding analytes, 1:LPMC1 and 2:LPMC2). When denatured andseparated from unbound probes, reaction components and sample material,the single-stranded ligation product molecular complexes (LPMC1 andLPMC2) are individually detected using at least one SMD. The order ofthe reporter groups is identified, indicating in this example that twospecies of analytes, i.e., 1 and 2, are present in the sample.

In certain embodiments, at least one analyte includes a nucleic acidsequence comprising at least one at least one deoxyribonucleotide, atleast one ribonucleotide, or both at least one deoxyribonucleotide andat least one ribonucleotide. In certain embodiments at least one analytecomprises a double-stranded nucleic acid sequence comprising DNA or RNA,such as genomic DNA, including but not limited to fragments such asrestriction enzyme fragments, shear fragments, or sonication-inducedfragments. In certain embodiments, at least one analyte comprises atleast one point mutation, at least one deletion, at least one insertion,at least one chromosomal translocation site, at least one splicejunction, or combinations thereof.

In certain embodiments, at least one analyte comprises a nucleic acidmolecule or a fragment thereof comprising at least one multi-alleliclocus. In certain embodiments, one or more multi-allelic locus comprisesat least one SNP. In certain embodiments, the disclosed compositions,methods, and kits allow one to determine which of two or more alternatesequences are present at a multi-allelic locus. In certain embodiments,a probe set comprises at least two different upstream probes, forexample but not limited to, allele-specific oligos (ASOs), and one atleast one downstream probe, for example but not limited to, alocus-specific oligonucleotide (LSO). In such probe sets, the at leasttwo upstream probes differ by at least one nucleotide in theirrespective reaction portions.

For example but without limitation, when analyzing the nucleic acid froman individual that is homozygous for a particular bi-allelic SNP, incertain embodiments, the reaction portion of only one upstream probe ofthe probe set will fully hybridize with the target sequence, while theother upstream probe will have at least one nucleotide in it's reactionportion that is not hybridized. Thus, under appropriate conditions, amolecular complex comprising only a single species of correspondingligation product will be formed, comprising the upstream probe with thefully complementary reaction portion ligated to the downstream probe,e.g., a LPMC. While two species of corresponding LPMCs will be formed,under appropriate conditions, when the nucleic acid sample is obtainedfrom a heterozygous individual. In certain embodiments, a probe setcomprises at least one upstream probe and at least two downstreamprobes. In such probe sets, the at least two downstream probes differ byat least one nucleotide in their respective reaction portions.

FIG. 2 schematically depicts an exemplary molecular complex fordetermining the presence of a nucleic acid analyte, for example but notlimited to, a nucleic acid sequence containing a multi-allelic locus,such as a SNP site. The exemplary molecular complex comprises an analytehybridized by its target sequence to the combined reaction portions ofthe ligation product. The exemplary ligation product molecular complexcomprises both an identity portion and an analytical portion. Theillustrative identity portion, comprises the ordered sequence ofreporter groups “FGFHHFFFFGF” and a reaction portion are shown on the 5′probe (“ASO” in FIG. 2). The analytical portion comprising reportergroup “I” and a reaction portion is shown on the 3′ probe of the ligatedprobe set (“LSO” in FIG. 2). The letter “X” indicates the SNP site onthe nucleic acid analyte and the ligation site is depicted by “Λ” inFIG. 2. The skilled artisan will understand that the identity portioncan be located, at least partially, in either the first probe or thesecond probe of a given probe set and that the analytical portion can belocated, at least partially, in either the first probe or the secondprobe of a given probe set, but typically, the entire identity portionand the entire analytical portion are not both located in the same probeof a given probe set.

In certain embodiments, at least one first probe and at least onecorresponding second probe hybridize to sequences on the same strand ofat least one analyte, at least one analyte surrogate, or both, but thefirst probe and the second probe are not hybridized adjacent to oneanother. In certain embodiments, at least one polymerase and at leastone ligation reagent are provided. In certain embodiments, underappropriate conditions, at least one polymerase can extend a hybridizedupstream probe by primer extension until the newly synthesized 3′ end ofthe upstream probe is adjacent to the 5′ end of the correspondingdownstream probe. In certain embodiments, the newly synthesized 3′ endof the upstream probe and the 5′ end of the downstream probe are ligatedtogether by at least one ligation agent to form a ligation productmolecular complex.

In certain embodiments, at least one nucleic acid analyte is amplifiedto generate at least one analyte surrogate. In one exemplary embodiment,shown in FIG. 6A, messenger RNA (mRNA) analytes (shown schematically asW. L, and M; each comprising a “poly A” tail) are amplified using primerextension with sequence specific primers (depicted as Pr1 and Pr2) togenerate single-stranded DNA analyte surrogates (depicted as ssDNA andW, L, or M but without poly A tails). Probe sets are added to the ssDNAanalyte surrogates and at least some first probes and at least somecorresponding second probes anneal with the corresponding analytesurrogates. The hybridized probe sets are ligated together in thepresence of at least one ligation agent and under appropriateconditions, forming three species of LPMC in this illustrativeembodiment, each comprising (i) an identity portion including a codedmolecular tag and (ii) an analytical portion that includes at least oneDNP moiety. The three exemplary LPMCs are placed on at least onesubstrate comprising a patterned array of anti-DNP antibody capturemoieties so that the molecular complexes are tethered to the substratevia the interaction between the anti-DNP antibody capture moieties andthe DNP capture ligands, as shown in FIG. 6A. The tethered molecularcomplexes are then individually detected using an appropriate SMDtechnique and the order of the reporter groups in each coded moleculartag is determined.

In another exemplary embodiment, shown in FIG. 6B, mRNA analytes areamplified, using in vitro translation, to generate translated analytesurrogates. These analyte surrogates are combined with probe sets, eachcomprising (i) specific polyclonal rabbit antibody comprising codedmolecular tags containing a biotin capture ligand, wherein the codedmolecular tag is attached to the antibody probe by a cleavable linkerand (ii) corresponding mouse IgG monoclonal antibody probes; andmolecular complexes form. The reaction mixture comprising molecularcomplexes is combined with a chromatography matrix comprising anti-mouseIgG antibodies and the unbound material is separated from the boundmolecular complexes comprising mouse IgG monoclonal antibodies. Thelinker is cleaved and cleavable components, i.e., the biotinylated codedmolecular tags are isolated. The isolated coded molecular tags arecombined with a streptavidin-coated substrate and the coded moleculartags are tethered to the substrate via biotin-streptavidin binding. Thetethered coded molecular tags are individually detected using anappropriate SMD technique and the order of the reporter groups isdetermined.

A variety of methods are available for obtaining nucleic acid sequences,such as genomic DNA, from biological samples that can be used with thedisclosed compositions, methods, and kits. Exemplary nucleic acidisolation techniques include (1) organic extraction followed by ethanolprecipitation, e.g., using a phenol/chloroform organic reagent (e.g.,Ausbel et al., eds., Current Protocols in Molecular Biology, John Wiley& Sons, New York (1995, including supplements through June 2003),preferably using an automated DNA extractor, e.g., the Model 341 DNAExtractor available from Applied Biosystems (Foster City, Calif.); (2)stationary phase adsorption methods (e.g., Boom et al., U.S. Pat. No.5,234,809; Walsh et al., BioTechniques 10(4): 506-513 (1991); and (3)salt-induced DNA precipitation methods (e.g., Miller et al., Nucl. AcidsRes., 16(3): 9-10 (1988)), such precipitation methods being typicallyreferred to as “salting-out” methods. In certain embodiments, wherein atleast one analyte comprises nucleic acid sequences, the above isolationmethods can further comprise an enzyme digestion step, e.g., digestionwith at least one proteolytic enzyme; and/or exposure to at least onesurfactant, such as at least one cationic detergent, at least onezwitterionic detergent, at least one anionic detergent, or combinationsthereof (see, e.g., Greenberg et al., U.S. patent application Ser. Nos.09/724,613 and U.S. Patent Application Number US 2002/0177139).Commercially available kits can be used to expedite such methods, forexample, Wizard® Genomic DNA Purification Kit and the RNAgents® TotalRNA Isolation System (both available from Promega, Madison, Wis.).Further, such methods have been automated or semi-automated using, forexample, the ABI PRISM™ 6700 Automated Nucleic Acid Workstation (AppliedBiosystems, Foster City, Calif.) or the ABI PRISM™ 6100 Nucleic AcidPrepStation and associated protocols, e.g., NucPrep™ Chemistry:Isolation of Genomic DNA from Animal and Plant Tissue, AppliedBiosystems Protocol 4333959 Rev. A (2002), Isolation of Total RNA fromCultured Cells, Applied Biosystems Protocol 4330254 Rev. A (2002); andABI PRISM™ Cell Lysis Control Kit, Applied Biosystems Protocol 4316607Rev. C (2001).

3. Non-Nucleic Acid Analytes.

The compositions, methods, and kits disclosed herein can also be used ina wide variety of applications to determine the presence of non-nucleicacid analytes in a sample. For example but without limitation, thecompositions, methods, and kits are useful for, pharmacokinetic studies,including but not limited to, drug metabolism, ADME profiling, andtoxicity studies; target validation for drug discovery; proteinexpression profiling; proteome analyses; metabolomic studies;post-translation modification studies, including but not limited toglycosylation, phosphorylation, acetylation, and amino acidmodification, such as modification of glutamate to form gamma-carboxyglutamate and hydroxylation of proline to form hydroxylation; analysesof specific serum or mucosal antibody levels; evaluation of non-nucleicacid diagnostic indicators; foreign antigen detection; and the like.

In certain embodiments, at least one analyte comprises at least oneamino acid, for example, a peptide or protein molecule; at least onecarbohydrate subunit, e.g. (—CHO—); at least one peptide bond; at leastone glycosidic bond; at least one fatty acid side chain; at least onealkyl group, allyl group, aryl group, and/or at least one aromatic ringstructure; or combinations thereof. In certain embodiments, at least oneprobe set comprises only first probes or only second probes, but notboth. In certain embodiments, at least one molecular complex comprisesat least one probe comprising at least one identity portion, but noseparate analytical portion, and the inherent properties of at least onemolecular complex serves as the basis for separating at least onemolecular complex, for example but not limited to, using capillaryelectrophoresis, gel filtration chromatography, HPLC, or the like (see,e.g., of FIG. 1B). In certain embodiments, at least one first probe orat least one second probe further comprises at least one cleavablecomponent, at least one cleavable linker, or both.

In certain embodiment, at least one first probe, at least one secondprobe, or both the first probes and the second probes of at least oneprobe set comprise at least one antibody that reacts specifically withat least one analyte or at least one analyte surrogate. In certainembodiments, at least one first probe, at least one corresponding secondprobe, or at least one first probe and at least one corresponding secondprobe, comprises at least one aptamer that reacts specifically with atleast one non-nucleic acid analyte or at least one analyte surrogate. Incertain embodiments, at least one first probe, at least one secondprobe, or both the first probes and the second probes of at least oneprobe set comprise binding proteins that specifically interact with atleast one analyte or at least one analyte surrogate.

The schematic in FIG. 1B depicts one exemplary embodiment comprising asample that includes non-nucleic acid analytes. Non-nucleic acidmolecules (shown as Protein 1, Protein 2, and Protein 3) and two singleprobe probe sets (shown as Probe 1 and Probe 2), each comprising ananalyte-specific antibody molecule comprising an identity portionattached with a cleavable crosslinker located between the reactionportion and the identity portion (Ab1-IP1 and Ab2-IP2, respectively),are combined and molecular complexes form (shown as MC1 and MC1). Noprobe set corresponding to Protein 3 is used, thus no molecular complexcomprising Protein 3 is formed. The molecular complexes are separatedusing, for example electrophoresis or chromatography, and the separatedmolecular complexes are treated with an appropriate reagent to cleavethe crosslinker and release cleavable components, each comprising anidentity portion (shown as IP1 and IP2). The cleavable components areindividually detected using an appropriate SMD technique and the orderof the reporter group species in the coded molecular tags is determined.

The skilled artisan understands that with antibody probes, the reactiveportion typically comprises the antigen binding site and relatedresidues of the antibody molecule; and the target sequences comprisethat portion of the analyte that includes the epitope, whether suchsequences are linear, conformational, or combinations thereof. Theskilled artisan will appreciate that the molecular complexes and the atleast part of the molecular complexes described herein can beindividually detected while tethered or attached to a substrate or whilein solution, depending on, among other things, the nature of thespecific molecular complex or cleavable component and the SMD techniqueand detection apparatus employed.

Protein isolation techniques are also well known in the art and kitsemploying at least some of these techniques are commercially available.Protein isolation techniques typically employ one or more of thefollowing: maceration and cell lysis, including physical, chemical andenzymatic methods; centrifugation; separations by molecular weight, suchas size exclusion chromatography and preparative electrophoresis;selective precipitation, for example, salting-in and salting-outprocedures; various chromatographic methods; and the like. Detaileddescriptions of and relevant protocols for protein purificationtechniques can be found in, among other places, Marchak et al.,Strategies for Protein Purification and Characterization: A LaboratoryCourse Manual, Cold Spring Harbor Press (1996); Essentials from Cells: ALaboratory Manual, D. Spector and R. Goldman, eds., Cold Spring HarborPress (2003); R. Simpson, Proteins and Proteomics: A Laboratory Manual,Cold Spring Harbor Press (2003); and D. Liebler, Introduction toProteomics, Humana Press (2002). Commercially available kits can also beused, for example but not limited to, ProteoExtract™ Partial ProteomeExtraction Kits (P-PEK) and ProteoExtract™ Complete Proteome ExtractionKits (C-PEK), available from CALBIOCHEM®, La Jolla, Calif. The skilledartisan will appreciate that non-nucleic acid analytes for use with theinventive compositions, methods, and kits can be readily obtainedwithout undue experimentation using such purification techniques andcommercial kits.

Expressly beyond the scope of the methods for determining the presenceof at least one analyte disclosed herein, are various polymer sequencingtechniques, for example but not limited to, DNA sequencing and proteinsequencing; and restriction enzyme mapping techniques. Such techniquesinclude, without limitation, cleaving identifiable subunits from one ormore polymer and detecting the cleaved subunits to determine thesequence of the polymer, e.g., Edman degradation and similar techniques;moving, relative to each other, (a) at least one polymer comprisingidentifiable subunits and (b)(i) at least one activation or excitationsource and (ii) at least one detector, to determine the sequence and/orstructure of the polymer, and similar techniques; and cleavingidentifiable fragments from at least one DNA sequence using one or morerestriction enzymes and measuring the size or length of the restrictionfragment and/or the shortened DNA polymer to generate a restriction mapfor the DNA, and similar techniques.

The invention, having been described above, may be better understood byreference to examples. The following examples are intended forillustration purposes only, and should not be construed as limiting thescope of the invention in any way.

EXAMPLE 1 Coded Molecular Tag Fabrication: Labeling Templates using PNAOpeners Comprising Reporter Groups

Six different PNA openers comprising at least one fluorescent reportergroup species (“FRG” in this example) are synthesized on an AB433APeptide Synthesizer (Applied Biosystems, Foster City, Calif.)essentially according to the manufacturer's instructions and knownmethods. Each of the six PNA openers comprise the sequence:FRG-OO-Lys-Lys-[core sequence 1]-OOO-[core sequence 2]-Lys-Lys, where 0refers to 8-amino-3,6-dioxaoctanoicacid linker, Lys refers to lysine, Jrefers to N-[2-aminoethyl-5-ylacetyl]isocytosine glycine, core sequence1 refers to the particular single-stranded DNA sequence that iscomplementary to a specific sequence on the full-length bacteriophagelambda genome (“λ-DNA” in this example), and core sequence 2 depends onthe sequence of core sequence 1, as shown. Table 1 shows the number ofthe illustrative PNA openers (“#”), the location of target sequence inλ-DNA (“Position”), the λ-DNA target sequence (“L-DNA Sequence”), thecorresponding first core sequence (“Core Sequence 1”), the correspondingsecond core sequence (“Core Sequence 2”), and the Sequence ID Number(SEQ ID NO.:) for the corresponding L-DNA Sequence, Core Sequence 1, andthe Core Sequence 2, respectively. TABLE 1 SEQ L-DNA Core Core ID #Position Sequence Sequence 1 Sequence 2 NO.: 1   105 GAAAAGAAAGCTTTCTTTTC JTTTTJTTTJ 3, 4, 5 2  4404 AGAGGAGGAG CTCCTCCTCT TJTJJTJJTJ6, 7, 8 3  8141 AAAGGAAAGG CCTTTCCTTT TTTJJTTTJJ 9, 10, 11 4 12460GGGAAGAGAG CTCTCTTCCC JJJTTJTJTJ 12, 13, 14 5 20727 AGAAAGGGGATCCCCTTTCT TJTTTJJJJT 15, 16, 17 6 25025 AGGAAGAAAA TTTTCTTCCTTJJTTJTTTT 18, 19, 20For simplicity, the FRGs are designated 1-6 to correspond to the PNAopener number. Thus, FRG-labeled PNA opener #1 comprises the sequence:FRG1OO-Lys-Lys-CTTTCTTTTC-OOO-JTTTTJTTTJ-Lys-Lys.

Two μg lambda DNA is digested with BstEII in 20 μL reaction buffer. Eachof the six FRG-labeled PNA openers (2-5 μM) are combined with the BstEIIdigested λ-DNA (0.1 μg/μL) in 10 μM NaHPO₄, pH 6.8 and incubated for atleast two hours at 37°-60° C. Following the incubation, the fabricatedcoded molecular tags comprising a six-labeling position λ-DNA templatewith the ordered FRG pattern of 123456 are isolated and stored for useas an “off-the-shelf” reagent for assembling analyte detection probes.Alternatively, the reporter group-labeled PNA openers can be synthesizedand stored for later use as “off-the-shelf” reagents.

The skilled artisan will appreciate that if, for example but withoutlimitation, position 105 is always labeled with the same reporter groupand that reporter group is not used in any other labeling position, theposition 105 reporter group can serve as an orientation point forindividually detecting such coded molecular tags in solution. Theskilled artisan understands that λ-DNA comprises many additionallabeling positions that could be used with corresponding PNA or pcPNAopeners. Additionally, PNA or pcPNA openers that have multiple bindingsites can be used to label multiple labeling sites if desired. Theskilled artisan also understands that, while six exemplary PNA openersand a λ-DNA template were used for illustration purposes in thisexample, coded molecular tags can be fabricated from a variety oftemplates and any of a number of template-specific PNA openers and/ortemplate-specific pcPNA openers. Further, the PNA and/or pcPNAcomponents of such coded molecular tags can comprise a number ofappropriate binding configurations, including without limitation,openers, clamps, and earring structures.

EXAMPLE 2 Coded Molecular Tag Fabrication: Restriction-LigationProcedure

Coded molecular tags were generated by recombinant techniques usingtemplates comprising genomic DNA from the bacteriophage lambda (λ-DNA)and two intercalating fluorescent dyes, as shown in FIG. 7A.

One microgram λ-DNA was combined with 10 units of the restriction enzymeNheI, bovine serum albumin (BSA) and 1×NEBuffer 2 in a reaction volumeof 20 μL and incubated at 37° C. for one hour (NheI RestrictionEndonuclease Kit, New England BioLabs, Beverly, Mass.). The restrictionenzyme digest was loaded onto a 0.7% agarose gel in 1×TBE andelectrophoresed at 1.5-2 volts/cm for 8 hours. Full-length (undigested)λ-DNA and a DNA ladder were electrophoresed in parallel as markers. Thegel was then stained with the intercalating dye SybrGreen (MolecularProbes, Eugene, Oreg.) and the stained material visualized under UVillumination. Full-length λ-DNA is a double stranded moleculeapproximately 48,500 base pairs (48.5 kilobase pairs (kb)) long.NheI-digested λ-DNA produced two restriction fragments, a smallerfragment of approximately 13 kb and a larger, 35 kb fragment. The bandscontaining the two restriction fragments were excised from the gel andthe fragments purified using a QIAEX II Gel Extraction kit according tothe manufacturer's protocol (Qiagen, Inc., Valencia, Calif.). Thepurified 13 kb and 35 kb fragments were stained for 1 hour in 1:10000dilutions of the intercalating dyes YOYO-1 or POPO-3, respectively(Molecular Probes, Inc.). These labeled coded molecular tag subunitswere spin column purified, then ligated together using T4 DNA ligaseaccording to the manufacturer's protocol (New England BioLabs). Suchcoded molecular tags can be individually detected using appropriate SMDtechniques, for example but not limited to, laser-confocal microscopy.

The skilled artisan will also understand that different coded moleculartags can be generated using the illustrative restriction fragmentsdescribed above, but labeled with different intercalating dyes, orlabeled in the opposite or a different order, i.e., the 13 kb fragmentlabeled with POPO-3 and the 35 kb fragment labeled with YOYO-1. Theskilled artisan will appreciate that different restriction fragments canbe generated using appropriate restriction enzymes and/or differentstarting materials without undue experimentation, using conventionalmethodology known in the art, for example without limitation, PCRamplified plasmids, as shown in FIG. 7B.

EXAMPLE 3 Coded Molecular Tag Fabrication.

Adenovirus-2 DNA (35.9 kb) is cleaved with Pac I (New England BioLabs#R0547) according to the manufacturer's instructions and the digestionproducts are gel purified using conventional methods. A 28.6 kb fragmentand a 7.3 kb fragment (“frag 1” in this example) are obtained. The 28.6kb fragment is cleaved with AsiS I (New England BioLabs #R0360)according to the manufacturer's instructions and the digestion productsare gel purified using conventional methods. A 21.4 kb fragment and a7.2 kb fragment (“frag 2” in this example) are obtained. The 21.4 kbfragment is cleaved with Pme I (New England Biolabs #R0560) according tothe manufacturer's instructions and the digestion products are gelpurified using conventional methods. A 13.2 kb fragment and an 8.2 kbfragment (“frag 3” in this example) are obtained. The 13.2 kb fragmentis cleaved with Sbf I (New England Biolabs #V0101) according to themanufacturer's instructions and the digestion products are gel purifiedusing conventional methods. An 8.4 kb fragment (“frag 4” in thisexample) and a 4.7 kb fragment are obtained. The four isolatedrestriction enzyme fragments are individually enzymatically-labeled withPacific Blue (frag 1), Oregon Green 488 (frag 2), Alexa Fluor 568 (frag3), and Alexa Fluor 660 (frag 4) using the ARES DNA Labeling Kits(Molecular Probes) and purified fluorophore labeled DNA fragmentsobtained. The labeled fragments are annealed, then ligated using theQuick Ligation™ kit (New England Biolabs #M2200S) to give a 31.2 kbcoded molecular tag comprising the ordered reporter group sequence:Pacific Blue-Oregon Green 488-Alexa Fluor 568-Alexa Fluor 660. Anoligonucleotide linker with the sequence:     GGCCGG....-3′ACGTCCGGCC....-5′ (SEQ ID NO.: 21)is synthesized using conventional phosphoramidite chemistry except thatinstead of thymidine phosphoramidite,5′-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2′-deoxyUridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Biotin dT, Glen Research Cat. No. 10-1038-xx, Sterling, Va.) is used.The resulting oligonucleotide comprises a biotin moiety, a cohesive endthat is compatible with an Sbf I cleavage site, an Hpa II restrictionsite, and an Hae III restriction site. Cleavage with Hpa II will resultin a 2 base pair (bp) cohesive end, while cleavage with Hae III causesblunt ends.

This synthetic oligonucleotide is annealed with the 31.2 kb codedmolecular tag, then ligated using the Quick Ligation™ kit. The resulting31.2 kb coded molecular tag-linker ligation product is purified usingconventional methods and stored for further use. When the 31.2 kb codedmolecular tag-linker ligation product is treated with Hpa II (NewEngland Biolabs #R0171), the sequence: CGG....-3′   C....-5′is removed from the linker portion of the ligation product, leaving a 2base pair (bp) overhang. Thus, an oligonucleotide comprising a reactionportion and the sequence “CG” at its 5′ end can anneal with the cleaved31.2 kb-oligo ligation product and under appropriate conditions, theoligonucleotide can be ligated with the cleaved 31.2 kb coded moleculartag-linker ligation product to assemble a probe comprising a reactionportion, a biotin moiety, and a coded molecular tag. This probe alsocomprises an Hae III restriction site between the biotin moiety and thereaction portion.

The skilled artisan will appreciate that if this exemplary probe iscleaved with the restriction enzyme Hae III under appropriateconditions, a cleavable component comprising a coded molecular tag and abiotin moiety will be released. The skilled artisan will also understandthat when combined with a substrate comprising avidin, streptavidin, orderivatives thereof, the cleavable component will become attached ortethered to the substrate via the biotin-avidin (i.e., captureligand-capture moiety) interaction. The tethered or attached cleavablecomponent can be individually detected using an appropriate SMDtechnique, for example but not limited to, laser-confocal microscopy,and the coded molecular tag can be decoded, i.e., the order of thereporter groups in the coded molecular tag are determined.

The skilled artisan will appreciate that the size and/or sequence of thelinker oligonucleotide can vary and that any desired restriction enzymesite can be incorporated, although typically not a cleavage site that ispresent in the coded molecular tag. The linker can be synthesized orprepared enzymatically and may or may not comprise at least one affinitytag that may or may not be cleavable (see, e.g., Soukop et al.,Bioconjug. Chem. 6:135-38 (1995); L. Klevan and G. Gebeyehu, Methods ofEnzymol. 184:561-77 (1990); Bioconjugate Techniques; M. Shimkus et al.,Proc. Natl. Acad. Sci. 82:2593-97 (1985); and K. Misiura et al., Nucl.Acids Res. 18:4345-54 (1990)). The skilled artisan will also appreciatethat the probe and the coded molecular tag sequence can be enzymaticallyattached or be crosslinked, for example but not limited to usingcleavable or non-cleavable chemical or photoaffinity crosslinkingagents.

EXAMPLE 4 Coded Molecular Tag Fabrication: Chemical Labeling ofRestriction Fragments

Lambda genomic DNA is cleaved with the restriction enzyme NheI and the35 and 14 kb fragments gel purified and isolated as described in Example2. The 14 kb fragment is placed in a microfuge tube (tube 1) on ice. The35 kb fragment is digested with the restriction enzyme XbaI according tothe manufacturer's protocol (New England Biolabs) and XbaI restrictionfragments of 24.5 and 10.2 kb are gel purified and isolated as describedin Example 2. The 10.2 kb fragment is placed in a microfuge tube (tube2) on ice and the 24.5 kb fragment is digested with the restrictionenzyme BsiWI according to the manufacturer's protocol (New EnglandBiolabs) and BsiWI restriction fragments of 5.2 and 19.3 kb are gelpurified and isolated as described in Example 2. The 5.2 kb fragment isplaced in a microfuge tube (tube 3) on ice and the 19.3 kb fragment isdigested with the restriction enzyme BsaI according to themanufacturer's protocol (New England Biolabs). The 7.9 and 11.4 kb BsaIrestriction fragments are gel purified and isolated as described inExample 2. The 7.9 and 11.4 kb fragment are placed in separate microfugetubes (tube 4 and tube 5, respectively) on ice.

The isolated restriction fragments are chemically-labeled using ULYSISNucleic Acid Labeling Kits (Molecular Probes) according to themanufacturer's protocol, except that the DNase I digestion step isomitted. For example, tubes 1 and 4 are separately labeled using theULYSIS kit with Pacific Blue fluorophores (catalog no. U-21658); tubes 2and 5 are separately labeled using the ULYSIS kit with Alexa Fluor 546fluorophores (catalog no. U-21652) and tube 3 is labeled using theULYSIS kit with Alexa Fluor 647 fluorophores (catalog no. U-21660).

The coded molecular tag subunits are relegated to form a coded moleculartag using the Quick Ligation™ Kit from New England Biolabs. The codedmolecular tag subunits in tube 5 are ligated to the labeled restrictionfragments in tube 4, and a 19.3 kb coded molecular tag is gel purifiedand isolated. This 19.3 ligation product is ligated to the labeledrestriction fragments in tube 3, and a 24.5 kb coded molecular tag isgel purified and isolated. This 24.5 kb coded molecular tag is ligatedto the coded molecular tag subunits in tube 2, and a 34.7 kb codedmolecular tag is gel purified and isolated. This 34.7 kb coded moleculartag is ligated to the coded molecular tag subunits in tube 1, and acoded molecular tag of approximately 48 kb is gel purified and isolated.Alternatively, all of the coded molecular tag subunits can be combinedand ligated in a single ligation step to generate a 48 kb codedmolecular tag. This 48 kb coded molecular tag, corresponding to fulllength A genomic DNA with the order Pacific Blue-Alexa Fluor 546-AlexaFluor 647-Pacific Blue-Alexa Fluor 546 can be used for probe assembly.

This coded molecular tag can be detected using, for example, a scanninglaser confocal microscope system, including a Nichia direct diode laser(˜405 nm), a double YAG (yttrium, aluminum, garnet) laser (˜555 nm), anda helium-neon laser (˜632 nm) in a single beam laser confocalconfiguration. Alternatively, a xenon arc lamp, filtered into threefavorable excitation lines, can be used as the illumination source toprovide a suitable fluorescent image for individual detection. Theskilled artisan understands that a wide variety of illumination sourcescan provide an acceptable fluorescent image and thus any suitabledetection method is within the scope of the invention.

The skilled artisan will appreciate that any two coded molecular tagsubunits can be used as coded molecular tags, not just the 48 kb codedmolecular tag; that the order of labels can be varied; that a variety ofdifferent reporter groups can used in fabricating coded molecular tags,for example, there are at least ten ULYSIS nucleic acid labeling kits,each with a different fluorophore; that coded molecular tags can befabricated using a wide variety of templates; and that one or moreappropriate adapter (e.g., oligonucleotide linker) can be added to oneor more ends of the coded molecular tag to facilitate probe assembly,e.g., for use as one or more interchangeable component in assembling theprobes disclosed herein.

EXAMPLE 5 Analysis of a Multi-allelic Locus: Amplification and SNPDetection

One form of hypercholesterolemia, referred to as familialhypercholesterolemia (FH), results from a SNP, identified as mutation“W23X” (131 G->A). To evaluate susceptibility to FH, one can bedetermine whether the “wild-type” or mutant form of the FH allele ispresent at the W23X SNP site.

Genomic DNA is obtained from a patient and, if desired, the gDNA can bePCR amplified using 5′ synthetic oligonucleotide primers with thesequence: ATAGACACAGGAAA (SEQ ID NO.: 22) and 3′ syntheticoligonucleotide primers with the sequence: GGGGAAACCCGTACTATACG (SEQ IDNO.: 23) using conventional methods known in the art. The analytes oramplicon analyte surrogates comprising the SNP sequence(s) of interest(“Amplicons” in this example) are combined with at least onecorresponding probe set.

The probe set comprises two species of upstream probe, referred to asASO1 and ASO2 in this example, and one species of downstream probe,referred to as LSO in this example. ASO1 is designed with a reactionportion comprising the sequence GCATCTCCTACAAGTG (SEQ ID NO.:24) and islabeled at its 5′ end with digoxigenin (DIG). ASO2 is designed with areaction portion comprising the sequence GCATCTCCTACMGTA (SEQ ID NO.:25)and is labeled at its 5′ end with 2,4-dinitrophenyl (DNP). ASO1 and ASO2probe species are synthesized on an ABI 3900 High-Throughput DNASynthesizer (Applied Biosystems, Foster City, Calif.) according toconventional methods. ASO1 is end-labeled using an aminolinkerphosphoramidite and then DIG-labeled using the DIG Oligonucleotide5′-End Labeling Set (Roche Diagnostics GmbH Cat. No. 1 480 863,Mannheim, Germany), essentially as described in the manufacturer'sinstructions. ASO2 is 5′ end-labeled with DNP-TEG phosphoramidite (GlenResearch Cat. No. 10-1985-95) essentially as described in themanufacturer's instructions and methods known in the art. Thecorresponding LSO probe species comprises the sequenceGGTCTGCGATGGATGGCC (SEQ ID NO.:26), wherein the first 12 nucleotides onthe 5′ end form the reaction portion, and the last four nucleotides onthe 3′ end can hybridize with an appropriate Apa I restriction fragment(“Oligo 5” in this example), and an identity portion.

The identity portion, comprising a nucleic acid coded molecular tag on aT7 bacteriophage template with an adapter, a commercially available ApaI linker (New England BioLabs Cat. #S1129S), ligated to the endcomprising the first (5′-most) base on the left end (see, e.g., T7restriction map, New England BioLabs 2002-2003 Catalog at page 320), isprepared. The coded molecular tag comprises fluorescent reporter groupsin the order Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 488, andAlexa Fluor 647, left to right. These identity portions are cleaved withApa I according to the manufacturer's instructions (New England BioLabs,Cat. #RO114S), then annealed with and ligated to copies of Oligo 5 underappropriate conditions to assemble probes of the exemplary probe set.

The three probe species of this exemplary probe set are combined withthe Amplicons and annealed, forming molecular complexes. Appropriateupstream probes are ligated to the downstream probes using the QuickLigation Kit (New England BioLabs) essentially as described in themanufacturer's instructions, forming ligation product molecularcomplexes. The reaction mixture, comprising the ligation productmolecular complexes, is heated and the ligated products are isolated andcombined with a substrate comprising a patterned surface includingevenly-spaced alternating lines of covalently bound, commerciallyavailable anti-DNP or anti-DIG antibody capture moieties (e.g., BethylLaboratories, Montgomery, Tex.; United States Biological, Swampscott,Mass.; ZYMED Laboratories, So. San Francisco, Calif.; Roche DiagnosticsGmbH, Penzberg, Germany). The alternating lines are typically spaced farenough apart that elongated molecular complexes do not overlap from oneline to the next, e.g., approximately 20 μm for full length λ-DNA. Theanti-DIG antibody capture moieties react immunospecifically withligation products comprising ASO1, while the anti-DNP antibody capturemoieties react immunospecifically with ligation products comprisingASO2, indirectly binding the corresponding ligation products to thesubstrate. The bound ligation products are elongated in a fluid flow andindividually detected using laser confocal microscopy. Detection of theordered reporter group Alexa Fluor 488-Alexa Fluor 568-Alexa Fluor488-Alexa Fluor 647 at a location corresponding to a line of anti-DIGantibody capture moieties indicates that the patient's gDNA comprisesthe “wild-type” sequence and is not susceptible to familialhypercholesterolemia. Detection of the ordered reporter group AlexaFluor 488-Alexa Fluor 568-Alexa Fluor 488-Alexa Fluor 647 at a locationcorresponding to a line of anti-DNP antibody capture moieties indicatesthat the patient's gDNA comprises the W23X mutation and the patient issusceptible to FH. Detection of the ordered reporter group Alexa Fluor488-Alexa Fluor 568-Alexa Fluor 488-Alexa Fluor 647 at both the anti-Digand anti-DNP locations indicates that the patient is heterozygous withrespect to this multiallelic locus.

The skilled artisan will appreciate that any number of multiallelic lociwith known SNP sequences can be evaluated using the compositions,methods, and kits described herein. The skilled artisan will alsoappreciate that many different types of capture ligands, correspondingcapture moieties, substrates, and identity portions can be employed withthe disclosed compositions, methods, and kits and that the location ofcapture ligands and identity portions can vary while keeping within thescope of the teachings herein.

EXAMPLE 6 p53 Mutation Analyses

A number of mutations in tumor suppressor genes, such as p53, have beenidentified in numerous human cancers (see, e.g., Ahrendt et al., Proc.Natl. Acad. Sci. USA 96:7382-87, 1996; de Cremoux et al., J. Natl.Cancer Inst. 91:641-43, 1999; Anderson et al., Radiat. Res. 154:473-76,2000; Kurose et al., Nature Genetics, 32:355-57, (2002); and Ohiro etal., Mol. Cell. Biol. 23:322-334, 2003). For example, but not limitedto, the wild type sequence for p53 as well as many known p53 mutationsare publicly available from numerous sources, such as the NationalCenter for Biotechnology Information (NCBI) “Entrez” web site(ncbi.nlm.nih.gov/Entrez), Japanese Patent No. JP 1998127300-A/6, andsunsite.unc.edu/dnam/mainpage (Cariello et al., Nucl. Acid Res.24:119-20, 1996).

Genomic DNA is isolated from a whole blood sample obtained from a breastcancer patient using conventional methods and/or commercially availablekits. The genomic DNA is combined with probe sets selected to identifythe presence or absence of three known p53 mutations observed inmedullary breast carcinoma, occurring at exon 7 codon 236 (“236”;TAC->TGC), exon 7 codon 248 (“248”; CGG->CAG), and exon 7 codon 252(“252”; deletion of codon 252 CTC)(see, e.g., P. deCremoux et al., J.Natl. Canc. Inst. 91:641-43 (1999)). The 236 probe set comprises twofirst probes with a 3′ sequences ending in “ . . . CMCTA” (236-1-1) and“CAACTG” (236-1-2) and a second probe comprising the sequence “CATGT . .. ” at the 5′ end (236-2). The 248 probe set comprises a first probecomprising the sequence “ . . . AACCG” at the 3′ end (248-1) and twosecond probes comprising the sequences “GGAGG . . . ” (248-2-1) and“AGAGG . . . ” (248-2-2) at their respective 5′ ends. The 252 probe setcomprises a first probe comprising the sequence “ . . . CTCAC” at its 5″end (252-1) and a second probe comprising the sequence “ . . . CCCAT” atits 3′ end (252-2). In this illustrative example, the breast cancerpatient carries the 236 point mutation, but not the 248 point mutationor the 252 deletion mutation.

The respective probes hybridize with the patient's genomic DNA underappropriate conditions and molecular complexes form. Taq ligase is addedand under appropriate conditions, ligation product molecular complexescomprising 236-1-1:236-2, 248-1:248-2-1, and 252-1:252-2 form. Eachligation product molecular complex further comprises at least oneaffinity portion comprising at least DNP capture ligand and at least oneidentity portion including a unique DNA coded molecular tag comprisingfluorescent reporter group species.

The ligation product molecular complexes are denatured and separated bycapillary electrophoresis. The molecular complexes are placed on amicroscope slide substrate coated with commercially available anti-DNPantibody capture moieties and incubated at room temperature to allowantibody binding. The substrate is washed to remove unbound components,then illuminated using a laser of appropriate excitation wavelength. Thefluorescent reporter groups in the coded molecular tags are individuallydetected using confocal microscopy with appropriate lasers, filters,etc. The order of reporter groups in each of the three coded moleculartags is identified and the presence of the p53 wild-type sequence atcodon 248, the wild-type sequence at codon 252 and the mutant sequenceat codon 237 is determined.

The skilled artisan understands that using the compositions, methods,and kits disclosed herein, heritable and somatic mutations can beanalyzed in single assay or multiplex reaction formats. The skilledartisan will appreciate that appropriate experimental conditions dependin part on the sequence of the probes being employed and the ligationagent, but that such reaction conditions are generally available or canbe calculated or experimentally determined without undue experimentationusing ordinary skill and techniques known in the art. The skilledartisan will also understand that amplification methods, including butnot limited to PCR or primer extension, can be employed to amplify lowcopy number nucleic acid analytes.

EXAMPLE 7 Nucleic Acid Amplification-Protein Detection

In one exemplary embodiment, mRNA analytes in a sample are amplified byin vitro translation, using a commercially available rabbit reticulocytelysate in vitro translation kit. As shown in FIG. 6B, mRNA analytesdesignated “1” and “2”, are amplified by in vitro translation to produceanalyte surrogates 1 and 2 (“AS1” and “AS2”). The two analyte surrogatesare combined with the corresponding probe sets. At least one first probeof each corresponding probe set comprises a rabbit polyclonal antibodyspecific for its corresponding antigen (“R1” and “R2”), a DNA codedmolecular tag comprising reporter groups 1, 2, and 3, at least onebiotin capture ligand within the coded molecular tag, and a cleavablelinker located between the antibody molecule and the identity portion.At least one second probe of each corresponding probe set comprises amouse IgG monoclonal antibody specific for its corresponding antigen(“M1” and “M2”). The skilled artisan will appreciate that the antibodiesfor each probe set are selected so that they bind to different,non-interfering, epitopes of the analyte or analyte surrogate than thecorresponding antibody.

The molecular complexes that form by the binding of the two antibodyprobes, are passed over a anti-mouse IgG sepharose column thatspecifically binds the second probes, separating the column boundmolecular complexes. The bound molecular complexes are washed usingappropriate buffer and the linker cleaved using an appropriate reagentto release cleavable components. These cleavable components, comprisingordered fluorescent reporter groups and at least one biotin captureligand at its proximal end, are collected and combined with a substratecomprising patterned streptavidin capture moieties (“SA”). The cleavablecomponents become indirectly tethered to the substrate by the binding ofat least one biotin capture ligand to at least one substrate-boundstreptavidin capture moiety. Due to the location of the at least onecapture ligand within the coded molecular tag, the identity portions aretethered to the substrate at the proximal end of the coded moleculartag, i.e., the end of the coded molecular tag that was closest to thecleavable linker of the intact first probe. Thus, when placed in anexternal field, such as a fluid flow or an electric field, the codedmolecular tag attachment point serves to orient the bar code, as shownin FIG. 5D. The substrate is illuminated with laser light of appropriateexcitation wavelength and the coded molecular tags are individuallydetected using confocal microscopy. The order of the fluorescentreporter groups in each coded molecular tag is identified, shown as 123and 213 in FIG. 6B, which correspond to mRNA analytes 1 and 2respectively. The skilled artisan will understand that a variety ofantibodies can be used in the methods of the invention, includingwithout limitation, polyclonal, monospecific, monoclonal, engineered,chimeric, humanized, FAb fragments, scFv fragments, and the like.

EXAMPLE 8 Foreign Antigen Detection

In certain embodiments, at least one analyte comprises at least oneforeign antigen, such the surface antigen of hepatitis B virus (HBsAg).There are four known subtypes of HBsAg, designated “adw”, “adr”, “ayw”and “ayr”. Thus, to determine if a patient is infected with a particularsubtype of hepatitis B virus, at least one probe set should include atleast one first probe, such as a monospecific polyclonal antibody, e.g.,an anti-peptide antibody, that binds to one common epitope on HBsAg, andat least four second probes, such as four different mouse monoclonalantibody species that each specifically binds to one of the four HBsAgsubtypes, i.e., anti-adw, anti-adr, anti-ayw, and anti-ayr, but don'tcross-react with the other subtypes or compete with the other probes.

In this example, the first probe comprises a rabbit polyclonalanti-HBsAg antibody comprising at least one biotin moiety (“b-1P”). Thecorresponding second probes comprise four different subtype-specificmonoclonal antibodies, each specifically binding a different HBsAgsubtype (“2Pdw”, “2Pdr”, “2Pyw”, and “2Pyr”, respectively) withoutaffecting the binding of b-1P, and vice versa. Each second probe furthercomprises an identity portion including a coded molecular tag with aninternal hybridization tag at the proximal end of the coded moleculartag (relative to the antibody molecule) and a cleavable linker grouplocated between the antibody molecule and the proximal end of the codedmolecular tag.

A sample comprising HBsAg of the adr subtype (“HB-adr”) is combined withthis illustrative probe set and incubated, allowing at least onemolecular complex, comprising b-1P:HB-adr:2Pdr, to form. CaptAvidinagarose gel (Molecular Probes Cat. # C-21386) is added to make a slurryand the biotinylated components, including the molecular complexes bind.The slurry is centrifuged in an Eppendorf bench top centrifuge to pelletthe agarose. The supernatant is discarded and the pellet is washed withphosphate-buffered saline, pH 7.0 (“PBS” in this example). The resultingpellet is re-suspended in an appropriate reagent to release thecleavable components comprising coded molecular tags, centrifuged, thesupernatant comprising the cleavable components is collected and dilutedin PBS or neutralized, depending on the cleavage reagent. Thesupernatant is combined with a substrate comprising at least onehybridization tag complement. The cleavable components become indirectlytethered to the substrate when the hybridization tag of the codedmolecular tag (capture ligand) hybridizes with its hybridization tagcomplement (capture moiety) on the substrate. A fluid flow is placedacross the surface of the substrate, stretching the coded molecular tagin the direction of flow from its tether. The substrate is illuminatedwith light of appropriate excitation wavelengths and the coded moleculartags are individually detected by laser confocal microscopy. The orderof fluorescent reporter groups is identified, allowing the presence ofHBsAg of the adr subtype in the sample to be determined.

EXAMPLE 9 Drug and Metabolite Detection

In this exemplary embodiment, the analytes phenyloin, an anti-convulsantdrug (“PHE” in this example); the arene oxide of phenyloin, an activeintermediate (“AOP” in this example); and 3-O-methylcatechol, a possibletoxic metabolite (“3OM” in this example); shown in FIG. 9, areidentified using antibodies and aptamers.

The nucleotide sequences of several custom nucleic acid aptamers, eachreactive with PHE, AOP, and 3OM, are obtained from a commercial source(e.g., RiNA GmbH, Berlin, Germany; SomaLogic, Boulder, Colo.).Alternatively, aptamers can be obtained, without undue experimentation,using the SELEX and anti-SELEX processes known in the art. Biotinylatedaptamers are prepared using conventional solid-phase synthesis using anApplied Biosystems 3400 DNA Synthesizer, appropriate nucleotidephosphoramidites, and biotin phosphoramidite (Glen Research Cat. No.10-1953-95) so that the aptamers are biotin labeled on their 3′-ends.The biotinylated aptamers are tested in a conventional binding assay toverify that they still bind to PHE, AOP, and 3OM after biotinylation.One reactive biotinylated aptamer is selected for use as a probe(“b-Apt” in this example).

Several monoclonal antibodies, each reactive with one of PHE, AOP, or3OM, but not cross-reactive with either of the other two compounds, aregenerated by and purchased from a custom antibody supplier (e.g.,Genemed Synthesis, Inc. So. San Francisco, Calif.; Biogenesis, Ltd.,Poole, UK; Fusion Antibodies, Ltd., Belfast, Northern Ireland). Themonoclonal antibodies are activated with the cleavableheterobifunctional crosslinker N-Succinimdyl3-(2-pyridyidithio)propionate (SPDP; Pierce Biotechnology Cat. No.21857), as described in Bioconjugate Techniques, particularly at page232, protocol steps 1-5.

The 5′ phosphate groups of three coded molecular tag species, eachcomprising a DNP capture ligand near the 3′ end (Coded molecular tag 1,Coded molecular tag 2, and Coded molecular tag 3 in this example), areseparately cystamine-modified using the crosslinker1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC; PierceBiotechnology Cat. No. 77149), as described in Bioconjugate Techniques,particularly at pages 651-52. The cystamine-modified coded moleculartags are combined with the activated monoclonal antibodies as follows:Coded molecular tag 1 with each of the PHE monoclonal antibodies; Codedmolecular tag 2 with each of the AOP monoclonal antibodies; and Codedmolecular tag 3 with each of the 3OM monoclonal antibodies; andconjugated essentially as described in Bioconjugate Techniques,particularly at pages 663-64 and Figure 407 (except that activatedantibody molecules are substituted for activated alkaline phosphatase inthe protocol) to assemble probes. Aliquots of the resulting codedmolecular tag-monoclonal antibody probes are tested to verify that theyretain immunoreactivity and appropriately reactive probes are selected(“Coded molecular tag 1-PHE”, “Coded molecular tag 2-AOP”, and “Codedmolecular tag 3-3OM”, respectively).

A whole blood sample is collected from a patient with epilepsy beingmedicated with Dilantin™ (phenyloin) and serum obtained usingconventional methods. The serum is placed in a Centrifree®micropartition device (Cat. No. 4104, Millipore Corp., Bedford, Mass.)and processed, essentially as described in the manufacturer'sinstructions, to obtain an ultrafiltrate (“Ultrafiltrate” in thisexample).

Probe sets comprising b-Apt and Coded molecular tag 1-PHE; b-Apt andCoded molecular tag 2-AOP; and b-Apt Coded molecular tag 3-3OM; arecombined with the Ultrafiltrate under conditions appropriate formolecular complex formation to occur. The reaction mixture comprisingthe molecular complexes is placed on a streptavidin-coated microscopeslide (Greiner Bio-One) and incubated at room temperature for thirtyminutes. The unbound material is removed and the slide is washed withPBS. The coded molecular tags are cleaved from the coded moleculartag-monoclonal antibody conjugates using dithiothreitol (DTT; PierceBiotechnology Cat. No. 20290), as described in Bioconjugate Techniques,particularly at pages 79-80, and the cleavable components comprising thecoded molecular tags are isolated. The isolated cleavable components arecombined with a substrate comprising anti-DNP antibody capture moietiesand the cleavable components become indirectly tethered to thesubstrate. The tethered cleavable components are individually detectedusing an appropriate single molecule detection technique and the orderof reporter groups are identified and quantified. The quantity of eachcoded molecular tag species allows the concentration of each of thethree analytes, i.e., PHE, AOP, and 3OM, to be determined.

EXAMPLE 10 Confocal Detection System

At least one molecular complex or at least part of a molecular complex,comprising a coded molecular tag in a low fluorescence buffer orsolvent, such as phosphate buffered saline, pH 8.0, Tris-EDTA buffer(TE), pH 8.0, or distilled de-ionized water is placed on a substrate, inthis example, a treated 1″×3″ quartz microscope slide (Technical GlassProducts, Inc., Painesville Twp., OH). At least one molecular complex orat least part of a molecular complex comprises a coded molecular tagcomprising λ DNA comprising the fluorescent reporter group species FAM™(488ex/520em), NED™ (488ex/570em) and Liz™ (488ex/660em). A treated1″×1″ quartz cover slip (Technical Glass Products, Inc.) coated with(3-aminopropyl)triethoxysilane (APTES) is placed over the slide so thatthe buffer comprising molecular complexes is between the slide and theAPTES-coated cover slip and the molecular complexes indirectly attachedto the slide. To further stretch or elongate the bound molecularcomplexes, the substrate can be placed in a directional flow or field,for example but not limited to a solution or agarose fluid flow, anelectric or dielectric field, or the like, so that at least onemolecular complex is stretched in the direction of flow or in the field(see, e.g., T. Perkins et al., Science 268:83-7 (1995); S. Matsuura etal., Nucl. Acids Res. 29(16):e79 (2001); D. Schwarz, U.S. Pat. No.6,294,136; and V. Namasivayam et al., Anal. Chem. 74:3378-85 (2002)).

Prior to use, the quartz slides and cover slips can be treated bysoaking in ethanol for 30 minutes with sonication, then water for 30minutes with sonication, then ethanol for an additional 30 minutes withsonication. Following the second ethanol/sonication step, the treatedslides and cover slips are ready for use or can be stored in distilleddeionized water.

As shown in FIG. 10, the slide (1) and cover slip (2) placed in astandard microscope slide holder mounted on a X-Y Piezo Flexure stage(P-517.2CL, Polytec PI, Germany). The stage is used for scanning thesubstrate and individually detecting the molecular complexes comprisingfluorescence reporter groups (3). The slide (1) is placed in the holderwith the cover slip (2) facing the illumination source. A multi-lineargon-ion laser (4) beam (488 nm, 514 nm) is passed through a488NB3/XLK06 laser line filter (5; Omega Optical Inc., Brattleboro, Vt.)to select the 488 nm line only, a neutral density filter to control thelaser intensity (6; Omega Optical Inc., Brattleboro, Vt.), and a 15×Galilean beam expander (7; Edmund Scientific, Barrington, N.J.), thenreflected towards the sample by an XF2037 (500DRLP)(Omega Optical Inc.,Brattleboro, Vt.) or a 500DCLP (Chroma Technology Corp., Rockingham,Vt.) dichroic longpass beam splitter (8). The beam is focused onto atleast one molecular complex (3) using a 40×/1.15NA (numerical aperture)water immersion objective lens (9; UAPO40XW3/340, Olympus Inc., Tokyo,Japan). The emitted fluorescence from the laser-illuminated molecularcomplexes on the substrate is collected by the objective lens (9),generating a collimated beam (10). The collimated beam (10) passesthrough the main dichroic longpass beam splitter (8), and is spectrallyseparated into three spectral channels (11, 12, 13) using two secondarydichroic filters (14; 540DRLP and 590DRLP, Omega Optical Inc.,Brattleboro, Vt.). In each of the three spectral channels, a bandpassfilter (15) is used to set the spectral range and further reduce theamount of laser light reaching the single photon counting detector (16).In this example, bandpass filters 520DF22, 570DF26, and 660DF14 (15;Omega Optical Inc., Brattleboro, Vt.) are used to produce spectral bandsof 520 nm FWHM 22 nm, 570 nm FWHM 26 nm, and 660 nm FWHM 14 nm,respectively.

The collimated beam in each channel is then focused by a 01LAO119Achromat 90 mm focal length tube lens (16; Melles Griot, Carlsbad,Calif.) onto a confocal pinhole comprising a SPCM-QC4 62.5 μm/0.27NAcore diameter fiber (17; PerkinElmer Optoelectronics, Canada). The lightexiting the fiber in each channel is collected by a separateSPCM-AQR-14-FC single photon counting detector (18; PerkinElmerOptoelectronics, Canada). Alternatively, instead of using a separatedetector for each spectral channel, an electron multiplying CCD cameramounted on a spectrograph can be used, for example but not limited to, aSensovation SamBa SE-34 camera (Ludwigshafen, Germany), mounted on aJobin-Yvon CP140-3301 spectrograph (Instruments SA, Inc. Edison N.J.).The detection system is controlled by and data collection performedusing software based on LabVIEW software (National Instruments, Austin,Tex.). A TTL (transistor-transistor logic) finite pulse train at a userselectable rate and duty cycle triggers analog output of voltages to theX and Y axes of the stage which in turn sets the scanning of themolecular complexes. A second TTL pulse train synchronized to the first(also at a user selectable rate and duty cycle) triggers analog input ofthe actual X and Y location and gates the single photon detectors tointegrate the photon count. The integrated photon signal from each ofthe three detectors is plotted against the actual X and Y locations forvisualization. The signal from each of the detectors is used fordetermining the presence and identity of the fluorescent reportergroups. The order of fluorescent reporter group species in eachindividually detected molecular complex is identified and the presenceof the corresponding analyte is determined.

The skilled artisan will appreciate that, while the confocal detectionsystem described herein is appropriate for certain SMD techniques, alarge number of detection systems can be used, as appropriate. Detaileddescriptions of exemplary SMD detection devices can be found in, amongother places, K. Weston et al., Anal. Chem. 74:5342-5349 (2002); H. Liet al., Anal. Chem. 75:1664-70 (2003); I. Braslavsky et al., Proc. Natl.Acad. Sci. 100:3960-64 (2003); N. Dovichi et al., Anal. Chem. 56:348-54(1984); M. Medina et al., BioEssays 24:758-64 (2002); J. Kim et al.,Anal. Chem. 73:5984-91 (2001); P. Tinnefeld et al., J. Phys. Chem.105:1989-8003 (2001); Z. Foldes-Papp et al., Proc. Natl. Acad. Sci.98:11509-14 (2001); Y. Ma et al., Electrophoresis 22:421-26 (2001); K.Swinney and D. Bornhop, Electrophoresis 21:1239-50 (2000); C. Seidel etal., U.S. Pat. No. 6,137,584; and D. Schwarz, U.S. Pat. No. 6,294,136.

EXAMPLE 11 Electrochemiluminescence Detection

Several probe species comprising reaction portions and cleavablecomponents including at least one capture ligand and a coded moleculartag comprising Ru(bpy)₃ ²⁺, Os(phen)₂(dppene)²⁺, and/or Al(HQS)₃ ³⁺ aresynthesized. The illustrative coded molecular tags comprise threelabeling positions, each occupied by. Probe sets are prepared comprisingone electrochemiluminescent reporter group-labeled first probe and acorresponding second probe comprising an analytical portion including amobility modifier (see, e.g., U.S. patent application Ser. No.09/522,640). When these probe sets are combined with correspondinganalytes, molecular complexes form.

The molecular complexes are separated using electrophoresis andisolated. The isolated molecular complexes are combined with anappropriate reagent to release the cleavable components, which areisolated. As shown in FIG. 11, the isolated cleavable components arecombined with a substrate comprising a conductive surface (110) with apatterned surface comprising appropriate capture moieties and matchedelectrodes (107-109), and a Ag/AgCl reference electrode (105). Thevarious electrodes can be selectively connected (101-103) to a powersource (104), such as a potentiometer, as shown. The cleavablecomponents are tethered to the surface of the substrate via captureligand-capture moiety interactions. A fluid flow, comprising 0.05 Mtripropylamine (TPA) in 0.1 M KH₂PO₄ is directed across the surface ofthe substrate, perpendicular to the electrode array, to elongate thebound cleavable components, as shown in FIG. 11 (fluid flow left toright). Typically, the pH of the solution is maintained between 6 and12.

A potential of 1.1 V (vs. the Ag/AgCl reference electrode) issequentially applied to the electrodes on the substrate, oxidizing theelectrochemiluminescent labels together with the co-reactant TPA andinitiating electrochemiluminescence. As each electrode is activated, amulti-channel SMD optical detection system comprising spectral channelsfor 620 nm, 584 nm, and 500 nm, is focused on a very small area of theelectrode surface so that on average only one cleavable component is inthe field of view (as shown in FIG. 11, switch 101 is closed, activatingelectrode 107, initiating ECL in the electrochemiluminescent reportergroup species in the cleavable components 106 tethered adjacent toelectrode 107). The order of the electrochemiluminescent reporter groupspecies in each individually detected cleavable component is identifiedand the presence of the corresponding analyte is determined.

The skilled artisan understands that a variety ofelectrochemiluminescent reporter groups can be employed in the disclosedcompositions, methods, and kits and individually detected as described.The skilled artisan also understands that other electrochemicalgeneration techniques and detection apparati can be employed toindividually detect electrochemiluminescent reporter groups in at leastone molecular complex, at least part of a molecular complex, or both.

EXAMPLE 12 Tethering and Attaching Coded Molecular Tags

Full-length λ-DNA comprising a multiplicity of reporter group species inan ordered pattern is end-labeled with biotin using conventional methods(“b-λ” in this example). The b-λ is suspended in distilled de-ionizedwater at a final concentration of 0.01 to 0.1 μg/mL. A streptavidincoated glass slide (Greiner Bio-One) is soaked in phosphate-bufferedsaline, pH 7.2 (“PBS” in this example), then blocked using a 1% solution(weight/volume) of bovine serum albumin (BSA) in PBS. The blocked slideis washed three times with PBS, then a hybridization chamber is attachedto the slide. The b-λ solution is introduced into the hybridizationchamber and incubated for two hours at 4° C., allowing the b-λ barcodesto become tethered to the streptavidin-coated slide. After theincubation, the slide is washed three times with PBS and is ready forindividual detection. The slide is then analyzed, using an appropriateSMD technique, to allow the attached λ-DNA molecules to be individuallydetected and the order of reporter group species in the correspondingcoded molecular tags to be identified.

Alternatively, a glass cover slip (VWR Scientific Products) is silanatedas follows. The glass slide is incubated in Piranha solution (70:30concentrated H₂SO₄ to H₂O₂) for 12 hours at room temperature. The coverslip is rinsed with deionized water, then incubated in a solution of 3%APTES in 95% ethanol for 1 hour. The cover slip is dipped in absoluteethanol and cured for one hour at 115° C. Next, the silanated cover slipis cooled to room temperature, then washed with 95% ethanol.

A drop of water comprising full-length λ DNA comprising a codedmolecular tag at a concentration of approximately 0.01-0.1 μg/mL isplaced on the silanated glass cover slip. An untreated glass slide isfloated on top, forcing the drop to spread to a thickness of a fewmicrons. The λ-DNA molecules comprising the coded molecular tags attachto the silanated cover slip and, as the air-water interface recedes dueto capillary action and evaporation, the λ-DNA molecules stretch andbecome elongated. The silanated cover slip is then analyzed, using anappropriate SMD technique, to allow the attached i-DNA molecules to beindividually detected and the order of reporter group species in thecorresponding coded molecular tags to be identified.

In yet another alternate method, λ-DNA comprising a coded molecular tagis suspended in a polymer solution (1-4% polyacrylamide in deionizedwater) at a concentration of 0.01-0.1 μg/mL. A glass cover slip isplaced in the holder and spun at 10,000-15,000 RPM. Alternately, a spincoating machine can be used. A small volume (0.5 μL) of the λ-DNApolymer solution is dropped onto the spinning cover slip and thesolution flows very rapidly towards the edges of the cover slip due tocentrifugal force. During this rapid radial flow, the λ-DNA in thepolymer solution experiences high shear force and stretch, elongatingthe DNA molecule. The flowing polymer solution dries very rapidly,effectively attaching the elongated λ-DNA molecules to the cover slip.The attached λ-DNA molecules are individually detected and the order ofreporter group species in the corresponding coded molecular tagsidentified using an appropriate SMD technique.

Detailed descriptions of additional molecular elongation methods can befound in, among other places, Yokota et al., Anal. Chem. 71:4418-22(1999); Bensimon et al., Science 265:2096-98 (1994); Smith et al.,Science 258:1122-26 (1992); and Perkins et al., Science 268:83-87(1995).

Although the invention has been described with reference to variousapplications, methods, and compositions, it will be appreciated thatvarious changes and modifications can be made without departing from theinvention. The foregoing examples are provided to better illustrate thedisclosed compositions, methods, and kits and are not intended to limitthe scope of the teachings herein.

1. A method for determining the presence of at least one analyte in asample, comprising: forming at least one molecular complex comprising(a) the at least one analyte and (b) at least one first probe comprisingat least one reaction portion and at least one identity portioncomprising at least one coded molecular tag; and individually detectingthe at least one molecular complex or at least part of the at least onemolecular complex to determine the presence of the at least one analytein the sample.
 2. The method of claim 1, wherein the molecular complexfurther comprises at least one second probe comprising at least onereaction portion and at least one analytical portion.
 3. The method ofclaim 1, wherein the at least one identity portion comprises amultiplicity of fluorescent reporter groups.
 4. The method of claim 3,wherein the at least one identity portion comprises at least one peptidenucleic acid (PNA), at least one pseudocomplementary peptide nucleicacid (pcPNA), or combinations thereof.
 5. The method of claim 1, whereinthe at least one identity portion comprises at least one affinity tag,at least one mobility modifier, or at least one affinity tag and atleast one mobility modifier.
 6. The method of claim 1, wherein the atleast one identity portion is within, coextensive with, or overlaps atleast part of the reaction portion.
 7. The method of claim 2, whereinthe at least one analytical portion is within, coextensive with, oroverlaps at least part of the reaction portion.
 8. The method of claim2, wherein the at least one analytical portion comprises at least onePNA, at least one pcPNA, at least one dendrimer, at least one reportergroup, or combinations thereof.
 9. The method of claim 2, wherein the atleast one analytical portion comprises at least one fluorophore, atleast one mobility modifier, at least affinity tag, or combinationsthereof.
 10. The method of claim 2, wherein at least part of thereaction portion of the at least one first probe and at least part ofthe reaction portion of the at least one second probe hybridize tocomplementary sequences on the same strand of the at least one analyte.11. The method of claim 10, wherein the at least part of the reactionportion of the at least one first probe and the at least part of thereaction portion of the at least one second probe hybridize adjacent toone another.
 12. The method of claim 11, further comprising at least oneligation agent.
 13. The method of claim 12, wherein the at least oneligation agent comprises at least one DNA ligase, at least one RNAligase, or combinations thereof.
 14. The method of claim 13, wherein theat least one DNA ligase, the at least one RNA ligase, or both the atleast one DNA ligase and the at least one RNA ligase comprises at leastone thermostable ligase.
 15. The method of claim 2, wherein the at leastone analyte comprises at least one nucleic acid sequence comprising atleast one ribonucleotide, at least one deoxyribonucleotide, or at leastone ribonucleotide and at least one deoxyribonucleotide.
 16. The methodof claim 15, wherein the at least one analyte comprises at least onenucleic acid sequence comprising at least one heritable mutation, atleast one somatic mutation, at least one single nucleotide polymorphism(SNP), at least one point mutation, at least one deletion mutation, atleast one insertion mutation, at least one chromosomal translocation, orcombinations thereof.
 17. The method of claim 1, wherein the at leastone analyte comprises at least one diagnostic indicator, at least oneforeign antigen, at least one drug, at least one metabolite, at leastone small molecule, at least one peptide, at least one nucleotide, atleast one glycosidic bond, or combinations thereof.
 18. The method ofclaim 2, wherein the at least one analyte comprises at least onediagnostic indicator, at least one foreign antigen, at least one drug,at least one metabolite, at least one small molecule, at least onepeptide, at least one nucleotide, at least one glycosidic bond, orcombinations thereof.
 19. The method of claim 1, wherein theindividually detecting comprises at least one scanning probe microscopytechnique, at least one applied optical spectroscopy technique, or bothat least one scanning probe microscopy technique and at least oneapplied optical spectroscopy technique.
 20. The method of claim 2,wherein the individually detecting comprises at least one scanning probemicroscopy technique, at least one applied optical spectroscopytechnique, or both at least one scanning probe microscopy technique andat least one applied optical spectroscopy technique.
 21. The method ofclaim 20, wherein the at least one scanning probe microscopy techniquecomprises atomic force microscopy (AFM), magnetic resonance forcemicroscopy (MRFM), scanning electrochemical microscopy (SECM), scanningtunneling microscopy, or combinations thereof.
 22. The method of claim20, wherein at least one applied optical spectroscopy techniquecomprises fluorescence correlation spectroscopy (FCS), evanescent waveinduced fluorescence spectroscopy (EWIFS), scanning near-field opticalmicroscopy (SNOM), surface enhances raman spectroscopy (SERS), surfaceenhanced resonant raman spectroscopy (SERRS), surface plasmon resonance(SPR), laser-confocal microscopy, or combinations thereof.
 23. Themethod of claim 20, wherein the individually detecting comprisesattaching at least one molecular complex or at least part of a molecularcomplex, tethering at least one molecular complex or at least part of amolecular complex, or both attaching and tethering at least onemolecular complex or at least part of a molecular complex directly orindirectly to a substrate.
 24. The method of claim 23, wherein thesubstrate comprises at least one capture moiety.
 25. The method of claim24, wherein the attaching, the tethering, or both the binding and thetethering comprises at least one first capture moiety and at least onesecond capture moiety, wherein the at least one first capture moiety andthe at least one second capture moiety are spatially separated from eachother on the substrate.
 26. The method of claim 3, wherein individuallydetecting comprises attaching at least one molecular complex or at leastpart of a molecular complex, tethering at least one molecular complex orat least part of a molecular complex, or both attaching and tethering atleast one molecular complex or at least part of a molecular complex to asubstrate and identifying the order of the fluorescent reporter groupsusing laser-confocal microscopy; and wherein the analyte comprises atleast one nucleic acid sequence comprising at least one ribonucleotide,at least one deoxyribonucleotide, or at least one ribonucleotide and atleast one deoxyribonucleotide; at least one heritable mutation, at leastone somatic mutation, at least one diagnostic indicator, at least oneforeign antigen, at least one drug, at least one metabolite, at leastone small molecule, or combinations thereof.
 27. The method of claim 3,wherein individually detecting comprises identifying the order ofreporter group species in a molecular complex or at least part of amolecular complex in solution using laser-confocal microscopy; andwherein the analyte comprises at least one nucleic acid sequencecomprising at least one ribonucleotide, at least onedeoxyribonucleotide, or at least one ribonucleotide and at least onedeoxyribonucleotide; at least one heritable mutation, at least onesomatic mutation, at least one diagnostic indicator, at least oneforeign antigen, at least one drug, at least one metabolite, at leastone small molecule, or combinations thereof.
 28. The method of claim 2,further comprising amplifying the molecular complex.
 29. A method fordetermining the presence of at least one analyte in a sample,comprising: amplifying at least one analyte to form at least one analytesurrogate, forming at least one molecular complex comprising (a) the atleast one analyte, at least one analyte surrogate, or at least oneanalyte and at least one analyte surrogate and (b) at least one firstprobe comprising at least one reaction portion and at least one identityportion comprising at least one coded molecular tag; and individuallydetecting the at least one molecular complex or at least part of atleast one molecular complex to determine the presence of the at leastone analyte in the sample.
 30. The method of claim 29, wherein themolecular complex further comprises at least one second probe comprisingat least one reaction portion and at least one analytical portion. 31.The method of claim 29, wherein the at least one identity portioncomprises a multiplicity of fluorescent reporter groups.
 32. The methodof claim 31, wherein the at least one identity portion comprises atleast one peptide nucleic acid (PNA), at least one pseudocomplementarypeptide nucleic acid (pcPNA), at least one dendrimer, or combinationsthereof.
 33. The method of claim 30, wherein the at least one identityportion further comprises at least one affinity tag, at least onemobility modifier, or at least one affinity tag and at least onemobility modifier.
 34. The method of claim 30, wherein the at least oneidentity portion is within, coextensive with, or overlaps at least partof the reaction portion.
 35. The method of claim 30, wherein the atleast one analytical portion is within, coextensive with, or overlaps atleast part of the reaction portion.
 36. The method of claim 30, whereinthe at least one analytical portion comprises at least one PNA, at leastone pcPNA, at least one reporter group, or combinations thereof.
 37. Themethod of claim 36, wherein the at least one analytical portioncomprises at least one fluorophore, at least one mobility modifier, atleast affinity tag, or combinations thereof.
 38. The method of claim 30,wherein at least part of the reaction portion of the at least one firstprobe and at least part of the reaction portion of the at least onesecond probe hybridize to complementary sequences on the same strand ofthe at least one analyte, the at least one analyte surrogate and the atleast one analyte or at least one analyte surrogate.
 39. The method ofclaim 38, wherein the at least part of the reaction portion of the atleast one first probe and the at least part of the reaction portion ofthe at least one second probe hybridize adjacent to one another.
 40. Themethod of claim 39, further comprising at least one ligation agent. 41.The method of claim 40, wherein the at least one ligation agentcomprises at least one DNA ligase, at least one RNA ligase, orcombinations thereof.
 42. The method of claim 41, wherein the at leastone DNA ligase, the at least one RNA ligase, or both the at least oneDNA ligase and the at least one RNA ligase comprises at least onethermostable ligase.
 43. The method of claim 30, wherein the at leastone analyte comprises at least one nucleic acid sequence comprising atleast one ribonucleotide, at least one deoxyribonucleotide, or at leastone ribonucleotide and at least one deoxyribonucleotide.
 44. The methodof claim 43, wherein the at least one analyte comprises at least onenucleic acid sequence comprising at least one heritable mutation, atleast one somatic mutation, or at least one heritable mutation and atleast one somatic mutation.
 45. The method of claim 30, wherein theindividually detecting comprises at least one scanning probe microscopytechnique, at least one applied optical spectroscopy technique, or bothat least one scanning probe microscopy technique and at least oneapplied optical spectroscopy technique.
 46. The method of claim 45,wherein the at least one scanning probe microscopy technique comprisesatomic force microscopy (AFM), magnetic resonance force microscopy(MRFM), scanning electrochemical microscopy (SECM), scanning tunnelingmicroscopy, or combinations thereof.
 47. The method of claim 45, whereinat least one applied optical spectroscopy technique comprisesfluorescence correlation spectroscopy (FCS), evanescent wave inducedfluorescence spectroscopy (EWIFS), scanning near-field opticalmicroscopy (SNOM), surface enhances raman spectroscopy (SERS), surfaceenhanced resonant raman spectroscopy (SERRS), surface plasmon resonance(SPR), laser-confocal microscopy, or combinations thereof.
 48. Themethod of claim 46, wherein the individually detecting comprisesattaching at least one molecular complex or at least part of a molecularcomplex, tethering at least one molecular complex or at least part of amolecular complex, or both attaching and tethering at least onemolecular complex or at least part of a molecular complex directly orindirectly to a substrate.
 49. The method of claim 48, wherein thesubstrate comprises at least one capture moiety.
 50. The method of claim49, wherein the attaching, the tethering, or both the binding and thetethering comprises at least one first capture moiety and at least onesecond capture moiety, wherein the at least one first capture moiety andthe at least one second capture moiety are spatially separated from eachother on the substrate.
 51. The method of claim 30, wherein individuallydetecting comprises attaching at least one molecular complex or at leastpart of a molecular complex, tethering at least one molecular complex orat least part of a molecular complex, or both attaching and tethering atleast one molecular complex or at least part of a molecular complexdirectly or indirectly to a substrate and individually detecting the atleast one molecular complex or at least part of a molecular complexusing laser-confocal microscopy; wherein at least one molecular complexcomprises a multiplicity of fluorescent reporter group species; andwherein the analyte comprises at least one nucleic acid sequencecomprising at least one ribonucleotide, at least onedeoxyribonucleotide, at least one heritable mutation, at least onesomatic mutation, at least one diagnostic indicator, at least oneforeign antigen, at least one drug, at least one metabolite, at leastone small molecule, or combinations thereof.
 52. The method of claim 31,wherein the at least one molecular complex or at least part of amolecular complex is individually detected in solution usinglaser-confocal microscopy; and wherein the analyte comprises at leastone nucleic acid sequence comprising at least one ribonucleotide, atleast one deoxyribonucleotide, at least one heritable mutation, at leastone somatic mutation, at least one diagnostic indicator, at least oneforeign antigen, at least one drug, at least one metabolite, at leastone small molecule, or combinations thereof.
 53. A kit for determiningthe presence of at least one analyte in a sample comprising at least oneprobe set comprising (a) at least one first probe comprising at leastone first reaction portion and (b) at least one second probe comprisingat least one second reaction portion, wherein at least one probe in theat least one probe set further comprises at least one identity portion,and wherein the at least one first probe and the at least one secondprobe are suitable for forming a molecular complex in the presence ofthe at least one analyte.
 54. The kit of claim 53, further comprising atleast one ligase, at least one polymerase, or at least one ligase and atleast one polymerase.
 55. The kit of claim 54, wherein the at least onepolymerase comprises at least one DNA polymerase, at least one RNApolymerase, at least one reverse transcriptase, or combinations thereof.56. The kit of claim 53, further comprising at least one dendrimer, atleast one PNA, at least one pcPNA, at least one intercalating substance,at least one antibody, or combinations thereof.
 57. The kit of claim 53,wherein at least one probe in the at least one probe set furthercomprises at least one analytical portion.
 58. The kit of claim 53,further comprising at least one reporter group.
 59. The kit of claim 53,further comprising biotin, a derivative of biotin, avidin, a derivativeof avidin, strepavidin, a derivative of streptavidin, or combinationsthereof.
 60. The kit of claim 59, further comprising at least onesubstrate.